Hepatitis C Virus Inhibitors

ABSTRACT

The present disclosure relates to compounds, compositions and methods for the treatment of hepatitis C virus (HCV) infection. Also disclosed are pharmaceutical compositions containing such compounds and methods for using these compounds in the treatment of HCV infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/467,602 filed Mar. 25, 2011 and U.S. Provisional Application Ser.No. 61/440,086 filed Feb. 7, 2011.

The present disclosure is generally directed to antiviral compounds, andmore specifically directed to compounds which can inhibit the functionof the NS5A protein encoded by Hepatitis C virus (HCV), compositionscomprising such compounds, and methods for inhibiting the function ofthe NS5A protein.

HCV is a major human pathogen, infecting an estimated 170 millionpersons worldwide—roughly five times the number infected by humanimmunodeficiency virus type 1. A substantial fraction of these HCVinfected individuals develop serious progressive liver disease,including cirrhosis and hepatocellular carcinoma.

The current standard of care for HCV, which employs a combination ofpegylated-interferon and ribavirin, has a non-optimal success rate inachieving sustained viral response and causes numerous side effects.Thus, there is a clear and long-felt need to develop effective therapiesto address this undermet medical need.

HCV is a positive-stranded RNA virus. Based on a comparison of thededuced amino acid sequence and the extensive similarity in the 5′untranslated region, HCV has been classified as a separate genus in theFlaviviridae family. All members of the Flaviviridae family haveenveloped virions that contain a positive stranded RNA genome encodingall known virus-specific proteins via translation of a single,uninterrupted, open reading frame.

Considerable heterogeneity is found within the nucleotide and encodedamino acid sequence throughout the HCV genome due to the high error rateof the encoded RNA dependent RNA polymerase which lacks a proof-readingcapability. At least six major genotypes have been characterized, andmore than 50 subtypes have been described with distribution worldwide.The clinical significance of the genetic heterogeneity of HCV hasdemonstrated a propensity for mutations to arise during monotherapytreatment, thus additional treatment options for use are desired. Thepossible modulator effect of genotypes on pathogenesis and therapyremains elusive.

The single strand HCV RNA genome is approximately 9500 nucleotides inlength and has a single open reading frame (ORF) encoding a single largepolyprotein of about 3000 amino acids. In infected cells, thispolyprotein is cleaved at multiple sites by cellular and viral proteasesto produce the structural and non-structural (NS) proteins. In the caseof HCV, the generation of mature non-structural proteins (NS2, NS3,NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. Thefirst one is believed to be a metalloprotease and cleaves at the NS2-NS3junction; the second one is a serine protease contained within theN-terminal region of NS3 (also referred to herein as NS3 protease) andmediates all the subsequent cleavages downstream of NS3, both in cis, atthe NS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B,NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiplefunctions by both acting as a cofactor for the NS3 protease andassisting in the membrane localization of NS3 and other viral replicasecomponents. The formation of a NS3-NS4A complex is necessary for properprotease activity resulting in increased proteolytic efficiency of thecleavage events. The NS3 protein also exhibits nucleoside triphosphataseand RNA helicase activities. NS5B (also referred to herein as HCVpolymerase) is a RNA-dependent RNA polymerase that is involved in thereplication of HCV genome with other HCV proteins, including NS5A, in areplicase complex.

Compounds useful for treating HCV-infected patients are desired whichselectively inhibit HCV viral replication. In particular, compoundswhich are effective to inhibit the function of the NS5A protein aredesired. The HCV NS5A protein is described, for example, in thefollowing references: S. L. Tan, et al., Virology, 284:1-12 (2001);K.-J. Park, et al., J. Biol. Chem., 30711-30718 (2003); T. L.Tellinghuisen, et al., Nature, 435, 374 (2005); R. A. Love, et al., J.Virol, 83, 4395 (2009); N. Appel, et al., J. Biol. Chem., 281, 9833(2006); L. Huang, J. Biol. Chem., 280, 36417 (2005); C. Rice, et al.,WO2006093867.

Bachand, et. al. in WO2008/021927, published Feb. 21, 2008, disclose aseries of biphenyl compounds which are useful for the treatment ofHepatitis C virus. The novel compounds of the present disclosure fallwithin the definition of the Formula in WO2008/021927 and are notdisclosed or described by Bachand, et al. Surprisingly, it has beendiscovered that these compounds possess unique attributes which makethem useful for the treatment of Hepatitis C virus.

In a first aspect the present disclosure provides a compound of Formula(I)

or a pharmaceutically acceptable salt thereof, wherein

R¹ is selected from hydrogen, methyl, and fluoro;

R² is selected from hydrogen and methyl;

R³ and R⁴ are each hydrogen; or

R³ and R⁴, together with the carbon atoms to which they are attached,form a cyclopropyl ring; and

R⁵ is selected from hydrogen and methyl.

In a first embodiment of the first aspect the present disclosureprovides a compound of Formula (I), or a pharmaceutically acceptablesalt thereof, wherein R¹ is hydrogen.

In a second embodiment of the first aspect the present disclosureprovides a compound of Formula (I), or a pharmaceutically acceptablesalt thereof, wherein R¹ is fluoro. In a third embodiment, R², R³, andR⁴ are each hydrogen and R⁵ is methyl.

In a fourth embodiment of the first aspect the present disclosureprovides a compound of Formula (I), or a pharmaceutically acceptablesalt thereof, wherein R¹ is methyl. In a fifth embodiment R², R³, and R⁴are each hydrogen and R⁵ is methyl.

In a second aspect the present disclosure provides a compound selectedfrom

or a pharmaceutically acceptable salt thereof

In a third aspect the present disclosure provides a compositioncomprising a compound of Formula (I), or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable carrier. In a firstembodiment of the third aspect the composition further comprises one,two, or three additional compounds having anti-HCV activity. In a secondembodiment of the third aspect at least one of the additional compoundsis an interferon or a ribavirin. In a third embodiment the interferon isselected from interferon alpha 2B, pegylated interferon alpha, consensusinterferon, interferon alpha 2A, interferon lambda, and lymphoblastiodinterferon tau.

In a fourth embodiment of the third aspect the present disclosureprovides a composition comprising a compound of Formula (I), or apharmaceutically acceptable salt thereof, a pharmaceutically acceptablecarrier, and one or two additional compounds having anti-HCV activity,wherein at least one of the additional compounds is selected frominterleukin 2, interleukin 6, interleukin 12, a compound that enhancesthe development of a type 1 helper T cell response, interfering RNA,anti-sense RNA, Imiqimod, ribavirin, an inosine 5′-monophospatedehydrogenase inhibitor, amantadine, and rimantadine.

In a fifth embodiment of the third aspect the present disclosureprovides a composition comprising a compound of Formula (I), or apharmaceutically acceptable salt thereof, a pharmaceutically acceptablecarrier, and one or two additional compounds having anti-HCV activity,wherein at least one of the additional compounds is effective to inhibitthe function of a target selected from HCV metalloprotease, HCV serineprotease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCVassembly, HCV egress, HCV NS5A protein, and IMPDH for the treatment ofan HCV infection.

In a fourth aspect the present disclosure provides a method of treatingan HCV infection in a patient, comprising administering to the patient atherapeutically effective amount of a compound of Formula (I), or apharmaceutically acceptable salt thereof. In a first embodiment of thefourth aspect the method further comprises administering one, two, orthree additional compounds having anti-HCV activity prior to, after orsimultaneously with the compound of Formula (I), or a pharmaceuticallyacceptable salt thereof. In a second embodiment of the fourth aspect atleast one of the additional compounds is an interferon or a ribavirin.In a third embodiment of the fourth aspect the interferon is selectedfrom interferon alpha 2B, pegylated interferon alpha, consensusinterferon, interferon alpha 2A, interferon lambda, and lymphoblastiodinterferon tau.

In a fourth embodiment of the fourth aspect the present disclosureprovides a method of treating an HCV infection in a patient, comprisingadministering to the patient a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof,and one or two additional compounds having anti-HCV activity prior to,after or simultaneously with the compound of Formula (I), or apharmaceutically acceptable salt thereof, wherein at least one of theadditional compounds is selected from interleukin 2, interleukin 6,interleukin 12, a compound that enhances the development of a type 1helper T cell response, interfering RNA, anti-sense RNA, Imiqimod,ribavirin, an inosine 5′-monophospate dehydrogenase inhibitor,amantadine, and rimantadine.

In a fifth embodiment of the fourth aspect the present disclosureprovides a method of treating an HCV infection in a patient, comprisingadministering to the patient a therapeutically effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof,and one or two additional compounds having anti-HCV activity prior to,after or simultaneously with the compound of Formula (I), or apharmaceutically acceptable salt thereof, wherein at least one of theadditional compounds is effective to inhibit the function of a targetselected from HCV metalloprotease, HCV serine protease, HCV polymerase,HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCV egress, HCVNS5A protein, and IMPDH for the treatment of an HCV infection.

In another aspect the present disclosure provides a compound which is

or a pharmaceutically acceptable salt thereof.

In another aspect the present disclosure provides a compound which is

or a pharmaceutically acceptable salt thereof.

Other embodiments of the present disclosure may comprise suitablecombinations of two or more of embodiments and/or aspects disclosedherein.

Yet other embodiments and aspects of the disclosure will be apparentaccording to the description provided below.

The compounds of the present disclosure also exist as tautomers;therefore the present disclosure also encompasses all tautomeric forms.

The description of the present disclosure herein should be construed incongruity with the laws and principals of chemical bonding.

It should be understood that the compounds encompassed by the presentdisclosure are those that are suitably stable for use as pharmaceuticalagent.

All patents, patent applications, and literature references cited in thespecification are herein incorporated by reference in their entirety. Inthe case of inconsistencies, the present disclosure, includingdefinitions, will prevail.

As used in the present specification, the following terms have themeanings indicated:

As used herein, the singular forms “a”, “an”, and “the” include pluralreference unless the context clearly dictates otherwise.

Asymmetric centers exist in the compounds of the present disclosure.These centers are designated by the symbols “R” or “S”, depending on theconfiguration of substituents around the chiral carbon atom. It shouldbe understood that the disclosure encompasses all stereochemicalisomeric forms, or mixtures thereof, which possess the ability toinhibit NS5A. Individual stereoisomers of compounds can be preparedsynthetically from commercially available starting materials whichcontain chiral centers or by preparation of mixtures of steroisomericproducts followed by separation such as conversion to a mixture ofdiastereomers followed by separation or recrystallization,chromatographic techniques, or direct separation on chiralchromatographic columns. Starting compounds of particularstereochemistry are either commercially available or can be made andresolved by techniques known in the art.

Certain compounds of the present disclosure may also exist in differentstable conformational forms which may be separable. Torsional asymmetrydue to restricted rotation about an asymmetric single bond, for examplebecause of steric hindrance or ring strain, may permit separation ofdifferent conformers. The present disclosure includes eachconformational isomer of these compounds and mixtures thereof.

The term “compounds of the present disclosure”, and equivalentexpressions, are meant to embrace compounds of Formula (I), andpharmaceutically acceptable enantiomers, diastereomers, and saltsthereof. Similarly, references to intermediates are meant to embracetheir salts where the context so permits.

The present disclosure is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include deuteriumand tritium. Isotopes of carbon include ¹³C and ¹⁴C.Isotopically-labeled compounds of the invention can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described herein, using an appropriateisotopically-labeled reagent in place of the non-labeled reagentotherwise employed. Such compounds may have a variety of potential uses,for example as standards and reagents in determining biologicalactivity. In the case of stable isotopes, such compounds may have thepotential to favorably modify biological, pharmacological, orpharmacokinetic properties.

The compounds of the present disclosure can exist as pharmaceuticallyacceptable salts. The term “pharmaceutically acceptable salt,” as usedherein, represents salts or zwitterionic forms of the compounds of thepresent disclosure which are water or oil-soluble or dispersible, whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of patients without excessive toxicity,irritation, allergic response, or other problem or complicationcommensurate with a reasonable benefit/risk ratio, and are effective fortheir intended use. The salts can be prepared during the final isolationand purification of the compounds or separately by reacting a suitablenitrogen atom with a suitable acid. Representative acid addition saltsinclude acetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate;digluconate, dihydrobromide, dihydrochloride, dihydroiodide,glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate,hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,lactate, maleate, mesitylenesulfonate, methanesulfonate,naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate,palmoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate,propionate, succinate, tartrate, trichloroacetate, trifluoroacetate,phosphate, glutamate, bicarbonate, para-toluenesulfonate, andundecanoate. Examples of acids which can be employed to formpharmaceutically acceptable addition salts include inorganic acids suchas hydrochloric, hydrobromic, sulfuric, and phosphoric, and organicacids such as oxalic, maleic, succinic, and citric.

When it is possible that, for use in therapy, therapeutically effectiveamounts of a compound of formula (I), as well as pharmaceuticallyacceptable salts thereof, may be administered as the raw chemical, it ispossible to present the active ingredient as a pharmaceuticalcomposition. Accordingly, the disclosure further provides pharmaceuticalcompositions, which include therapeutically effective amounts ofcompounds of formula (I) or pharmaceutically acceptable salts thereof,and one or more pharmaceutically acceptable carriers, diluents, orexcipients. The term “therapeutically effective amount,” as used herein,refers to the total amount of each active component that is sufficientto show a meaningful patient benefit, e.g., a reduction in viral load.When applied to an individual active ingredient, administered alone, theterm refers to that ingredient alone. When applied to a combination, theterm refers to combined amounts of the active ingredients that result inthe therapeutic effect, whether administered in combination, serially,or simultaneously. The compounds of formula (I) and pharmaceuticallyacceptable salts thereof, are as described above. The carrier(s),diluent(s), or excipient(s) must be acceptable in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof. In accordance with another aspectof the present disclosure there is also provided a process for thepreparation of a pharmaceutical formulation including admixing acompound of formula (I), or a pharmaceutically acceptable salt thereof,with one or more pharmaceutically acceptable carriers, diluents, orexcipients. The term “pharmaceutically acceptable,” as used herein,refers to those compounds, materials, compositions, and/or dosage formswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of patients without excessive toxicity,irritation, allergic response, or other problem or complicationcommensurate with a reasonable benefit/risk ratio, and are effective fortheir intended use.

Pharmaceutical formulations may be presented in unit dose formscontaining a predetermined amount of active ingredient per unit dose.Dosage levels of between about 0.01 and about 250 milligram per kilogram(“mg/kg”) body weight per day, preferably between about 0.05 and about100 mg/kg body weight per day of the compounds of the present disclosureare typical in a monotherapy for the prevention and treatment of HCVmediated disease. Typically, the pharmaceutical compositions of thisdisclosure will be administered from about 1 to about 5 times per day oralternatively, as a continuous infusion. Such administration can be usedas a chronic or acute therapy. The amount of active ingredient that maybe combined with the carrier materials to produce a single dosage formwill vary depending on the condition being treated, the severity of thecondition, the time of administration, the route of administration, therate of excretion of the compound employed, the duration of treatment,and the age, gender, weight, and condition of the patient. Preferredunit dosage formulations are those containing a daily dose or sub-dose,as herein above recited, or an appropriate fraction thereof, of anactive ingredient. Treatment may be initiated with small dosagessubstantially less than the optimum dose of the compound. Thereafter,the dosage is increased by small increments until the optimum effectunder the circumstances is reached. In general, the compound is mostdesirably administered at a concentration level that will generallyafford antivirally effective results without causing any harmful ordeleterious side effects.

When the compositions of this disclosure comprise a combination of acompound of the present disclosure and one or more additionaltherapeutic or prophylactic agent, both the compound and the additionalagent are usually present at dosage levels of between about 10 to 150%,and more preferably between about 10 and 80% of the dosage normallyadministered in a monotherapy regimen.

Pharmaceutical formulations may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, nasal, topical (including buccal, sublingual, ortransdermal), vaginal, or parenteral (including subcutaneous,intracutaneous, intramuscular, intra-articular, intrasynovial,intrasternal, intrathecal, intralesional, intravenous, or intradermalinjections or infusions) route. Such formulations may be prepared by anymethod known in the art of pharmacy, for example by bringing intoassociation the active ingredient with the carrier(s) or excipient(s).Oral administration or administration by injection are preferred.

Pharmaceutical formulations adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilemulsions.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water, and the like. Powders are prepared by pulverizing thecompound to a suitable fine size and mixing with a similarly comminutedpharmaceutical carrier such as an edible carbohydrate, as, for example,starch or mannitol. Flavoring, preservative, dispersing, and coloringagent can also be present.

Capsules are made by preparing a powder mixture, as described above, andfilling formed gelatin sheaths. Glidants and lubricants such ascolloidal silica, talc, magnesium stearate, calcium stearate, or solidpolyethylene glycol can be added to the powder mixture before thefilling operation. A disintegrating or solubilizing agent such asagar-agar, calcium carbonate, or sodium carbonate can also be added toimprove the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants,disintegrating agents, and coloring agents can also be incorporated intothe mixture. Suitable binders include starch, gelatin, natural sugarssuch as glucose or beta-lactose, corn sweeteners, natural and syntheticgums such as acacia, tragacanth or sodium alginate,carboxymethylcellulose, polyethylene glycol, and the like. Lubricantsused in these dosage forms include sodium oleate, sodium chloride, andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, betonite, xanthan gum, and the like. Tablets areformulated, for example, by preparing a powder mixture, granulating orslugging, adding a lubricant and disintegrant, and pressing intotablets. A powder mixture is prepared by mixing the compound, suitablecomminuted, with a diluent or base as described above, and optionally,with a binder such as carboxymethylcellulose, an aliginate, gelating, orpolyvinyl pyrrolidone, a solution retardant such as paraffin, aresorption accelerator such as a quaternary salt and/or and absorptionagent such as betonite, kaolin, or dicalcium phosphate. The powdermixture can be granulated by wetting with a binder such as syrup, starchpaste, acadia mucilage, or solutions of cellulosic or polymericmaterials and forcing through a screen. As an alternative togranulating, the powder mixture can be run through the tablet machineand the result is imperfectly formed slugs broken into granules. Thegranules can be lubricated to prevent sticking to the tablet formingdies by means of the addition of stearic acid, a stearate salt, talc, ormineral oil. The lubricated mixture is then compressed into tablets. Thecompounds of the present disclosure can also be combined with a freeflowing inert carrier and compressed into tablets directly without goingthrough the granulating or slugging steps. A clear or opaque protectivecoating consisting of a sealing coat of shellac, a coating of sugar orpolymeric material, and a polish coating of wax can be provided.Dyestuffs can be added to these coatings to distinguish different unitdosages.

Oral fluids such as solution, syrups, and elixirs can be prepared indosage unit form so that a given quantity contains a predeterminedamount of the compound. Syrups can be prepared by dissolving thecompound in a suitably flavored aqueous solution, while elixirs areprepared through the use of a non-toxic vehicle. Solubilizers andemulsifiers such as ethoxylated isostearyl alcohols and polyoxyethylenesorbitol ethers, preservatives, flavor additive such as peppermint oilor natural sweeteners, or saccharin or other artificial sweeteners, andthe like can also be added.

Where appropriate, dosage unit formulations for oral administration canbe microencapsulated. The formulation can also be prepared to prolong orsustain the release as for example by coating or embedding particulatematerial in polymers, wax, or the like.

The compounds of formula (I), and pharmaceutically acceptable saltsthereof, can also be administered in the form of liposome deliverysystems, such as small unilamellar vesicles, large unilamellar vesicles,and multilamellar vesicles. Liposomes can be formed from a variety ofphopholipids, such as cholesterol, stearylamine, or phophatidylcholines.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be delivered by the use of monoclonal antibodies asindividual carriers to which the compound molecules are coupled. Thecompounds may also be coupled with soluble polymers as targetable drugcarriers. Such polymers can include polyvinylpyrrolidone, pyrancopolymer, polyhydroxypropylmethacrylamidephenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysinesubstituted with palitoyl residues. Furthermore, the compounds may becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathicblock copolymers of hydrogels.

Pharmaceutical formulations adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active ingredient may be delivered from the patch byiontophoresis as generally described in Pharmaceutical Research 1986,3(6), 318.

Pharmaceutical formulations adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols, or oils.

Pharmaceutical formulations adapted for rectal administration may bepresented as suppositories or as enemas.

Pharmaceutical formulations adapted for nasal administration wherein thecarrier is a solid include a course powder having a particle size forexample in the range 20 to 500 microns which is administered in themanner in which snuff is taken, i.e., by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid, foradministration as a nasal spray or nasal drops, include aqueous or oilsolutions of the active ingredient.

Pharmaceutical formulations adapted for administration by inhalationinclude fine particle dusts or mists, which may be generated by means ofvarious types of metered, dose pressurized aerosols, nebulizers, orinsufflators.

Pharmaceutical formulations adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams, or sprayformulations.

Pharmaceutical formulations adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats, and soutes which renderthe formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders,granules, and tablets.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations may include other agents conventionalin the art having regard to the type of formulation in question, forexample those suitable for oral administration may include flavoringagents.

The term “patient” includes both human and other mammals.

The term “treating” refers to: (i) preventing a disease, disorder orcondition from occurring in a patient that may be predisposed to thedisease, disorder, and/or condition but has not yet been diagnosed ashaving it; (ii) inhibiting the disease, disorder, or condition, i.e.,arresting its development; and (iii) relieving the disease, disorder, orcondition, i.e., causing regression of the disease, disorder, and/orcondition.

The compounds of the present disclosure can also be administered with acyclosporin, for example, cyclosporin A or other analogs working throughsimilar mechanism. Cyclosporin A has been shown to be active against HCVin clinical trials (Hepatology 2003, 38, 1282; Biochem. Biophys. Res.Commun. 2004, 313, 42; J. Gastroenterol. 2003, 38, 567).

Table 1 below lists some illustrative examples of compounds that can beadministered with the compounds of this disclosure. The compounds of thedisclosure can be administered with other anti-HCV active compounds incombination therapy, either jointly or separately, or by combining thecompounds into a composition.

TABLE 1 Physiological Type of Inhibitor or Brand Name Class TargetSource Company NIM811 Cyclophilin Inhibitor Novartis ZadaxinImmuno-modulator Sciclone Suvus Methylene blue Bioenvision Actilon TLR9agonist Coley (CPG10101) Batabulin (T67) Anticancer β-tubulin inhibitorTularik Inc., South San Francisco, CA ISIS 14803 Antiviral antisenseISIS Pharmaceuticals Inc, Carlsbad, CA/Elan Phamaceuticals Inc., NewYork, NY Summetrel Antiviral antiviral Endo Pharmaceuticals HoldingsInc., Chadds Ford, PA GS-9132 (ACH- Antiviral HCV InhibitorAchillion/Gilead 806) Pyrazolopyrimidine Antiviral HCV Inhibitors ArrowTherapeutics compounds and salts Ltd. From WO-2005047288 26 May 2005Levovirin Antiviral IMPDH inhibitor Ribapharm Inc., Costa Mesa, CAMerimepodib Antiviral IMPDH inhibitor Vertex (VX-497) PharmaceuticalsInc., Cambridge, MA XTL-6865 (XTL- Antiviral monoclonal antibody XTL002) Biopharmaceuticals Ltd., Rehovot, Isreal Telaprevir Antiviral NS3serine protease Vertex (VX-950, LY- inhibitor Pharmaceuticals 570310)Inc., Cambridge, MA/Eli Lilly and Co. Inc., Indianapolis, IN HCV-796Antiviral NS5B Replicase Wyeth/Viropharma Inhibitor NM-283 AntiviralNS5B Replicase Idenix/Novartis Inhibitor GL-59728 Antiviral NS5BReplicase Gene Labs/ Inhibitor Novartis GL-60667 Antiviral NS5BReplicase Gene Labs/ Inhibitor Novartis 2′C MeA Antiviral NS5B ReplicaseGilead Inhibitor PSI 6130 Antiviral NS5B Replicase Roche Inhibitor R1626Antiviral NS5B Replicase Roche Inhibitor 2′C Methyl Antiviral NS5BReplicase Merck adenosine Inhibitor JTK-003 Antiviral RdRp inhibitorJapan Tobacco Inc., Tokyo, Japan Levovirin Antiviral ribavirin ICNPharmaceuticals, Costa Mesa, CA Ribavirin Antiviral ribavirinSchering-Plough Corporation, Kenilworth, NJ Viramidine AntiviralRibavirin Prodrug Ribapharm Inc., Costa Mesa, CA Heptazyme Antiviralribozyme Ribozyme Pharmaceuticals Inc., Boulder, CO BILN-2061 Antiviralserine protease Boehringer inhibitor Ingelheim Pharma KG, Ingelheim,Germany SCH 503034 Antiviral serine protease Schering Plough inhibitorZadazim Immune modulator Immune modulator SciClone Pharmaceuticals Inc.,San Mateo, CA Ceplene Immunomodulator immune modulator MaximPharmaceuticals Inc., San Diego, CA CellCept Immunosuppressant HCV IgGimmuno- F. Hoffmann-La suppressant Roche LTD, Basel, Switzerland CivacirImmunosuppressant HCV IgG immuno- Nabi suppressant BiopharmaceuticalsInc., Boca Raton, FL Albuferon - α Interferon albumin IFN-α2b HumanGenome Sciences Inc., Rockville, MD Infergen A Interferon IFN InterMunealfacon-1 Pharmaceuticals Inc., Brisbane, CA Omega IFN Interferon IFN-ωIntarcia Therapeutics IFN-β and Interferon IFN-β and EMZ701 TransitionEMZ701 Therapeutics Inc., Ontario, Canada Rebif Interferon IFN-β1aSerono, Geneva, Switzerland Roferon A Interferon IFN-α2a F. Hoffmann-LaRoche LTD, Basel, Switzerland Intron A Interferon IFN-α2bSchering-Plough Corporation, Kenilworth, NJ Intron A and InterferonIFN-α2b/α1-thymosin RegeneRx Zadaxin Biopharma. Inc., Bethesda, MD/SciClone Pharmaceuticals Inc, San Mateo, CA Rebetron InterferonIFN-α2b/ribavirin Schering-Plough Corporation, Kenilworth, NJ ActimmuneInterferon INF-γ InterMune Inc., Brisbane, CA Interferon-β InterferonInterferon-β-1a Serono Multiferon Interferon Long lasting IFN Viragen/Valentis Wellferon Interferon Lympho-blastoid IFN- GlaxoSmithKline αn1plc, Uxbridge, UK Omniferon Interferon natural IFN-α Viragen Inc.,Plantation, FL Pegasys Interferon PEGylated IFN-α2a F. Hoffmann-La RocheLTD, Basel, Switzerland Pegasys and Interferon PEGylated IFN-α2a/ MaximCeplene immune modulator Pharmaceuticals Inc., San Diego, CA Pegasys andInterferon PEGylated IFN- F. Hoffmann-La Ribavirin α2a/ribavirin RocheLTD, Basel, Switzerland PEG-Intron Interferon PEGylated IFN-α2bSchering-Plough Corporation, Kenilworth, NJ PEG-Intron/ InterferonPEGylated IFN- Schering-Plough Ribavirin α2b/ribavirin Corporation,Kenilworth, NJ IP-501 Liver protection antifibrotic IndevusPharmaceuticals Inc., Lexington, MA IDN-6556 Liver protection caspaseinhibitor Idun Pharmaceuticals Inc., San Diego, CA ITMN-191 (R-7227)Antiviral serine protease InterMune inhibitor Pharmaceuticals Inc.,Brisbane, CA GL-59728 Antiviral NS5B Replicase Genelabs InhibitorANA-971 Antiviral TLR-7 agonist Anadys Boceprevir Antiviral serineprotease Schering Plough inhibitor TMS-435 Antiviral serine proteaseTibotec BVBA, inhibitor Mechelen, Belgium BI-201335 Antiviral serineprotease Boehringer inhibitor Ingelheim Pharma KG, Ingelheim, GermanyMK-7009 Antiviral serine protease Merck inhibitor PF-00868554 Antiviralreplicase inhibitor Pfizer ANA598 Antiviral Non-Nucleoside Anadys NS5BPolymerase Pharmaceuticals, Inhibitor Inc., San Diego, CA, USA IDX375Antiviral Non-Nucleoside Idenix Replicase Inhibitor Pharmaceuticals,Cambridge, MA, USA BILB 1941 Antiviral NS5B Polymerase BoehringerInhibitor Ingelheim Canada Ltd R&D, Laval, QC, Canada PSI-7851 AntiviralNucleoside Pharmasset, Polymerase Inhibitor Princeton, NJ, USA PSI-7977Antiviral Nucleotide NS5B Pharmasset, Polymerase Inhibitor Princeton,NJ, USA VCH-759 Antiviral NS5B Polymerase ViroChem Pharma InhibitorVCH-916 Antiviral NS5B Polymerase ViroChem Pharma Inhibitor GS-9190Antiviral NS5B Polymerase Gilead Inhibitor Peg-interferon AntiviralInterferon ZymoGenetics/ lamda Bristol-Myers Squibb INX-189 AntiviralNucleotide NS5B Inhibitex Polymerase Inhibitor

The compounds of the present disclosure may also be used as laboratoryreagents. Compounds may be instrumental in providing research tools fordesigning of viral replication assays, validation of animal assaysystems and structural biology studies to further enhance knowledge ofthe HCV disease mechanisms. Further, the compounds of the presentdisclosure are useful in establishing or determining the binding site ofother antiviral compounds, for example, by competitive inhibition.

The compounds of this disclosure may also be used to treat or preventviral contamination of materials and therefore reduce the risk of viralinfection of laboratory or medical personnel or patients who come incontact with such materials, e.g., blood, tissue, surgical instrumentsand garments, laboratory instruments and garments, and blood collectionor transfusion apparatuses and materials.

This disclosure is intended to encompass compounds having formula (I)when prepared by synthetic processes or by metabolic processes includingthose occurring in the human or animal body (in vivo) or processesoccurring in vitro.

The abbreviations used in the present application, includingparticularly in the examples which follow, are well-known to thoseskilled in the art. Some of the abbreviations used are as follows: h,hr, or hrs for hours; EtOAc for ethyl acetate; Hex for hexanes; DCM fordichloromethane; DEAD for diethyl azodicarboxylate; Ph₃P fortriphenylphosphine; Et₂O for diethyl ether; THF for tetrahydrofuran;LiHMDS for lithium hexamethyldisilazide; Ph for phenyl; DIEA or DIPEA oriPr₂EtN for diiosopropylethylamine; EtOH for ethanol; MeOH for methanol;DMSO for dimethylsulfoxide; RT or Rt or rt or Rt for room temperature orretention time (context will dictate); ON or o/n for overnight; min forminutes; DCM for dichloromethane; HATU forO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate; DMF for N,N-dimethylformamide; TFA fortrifluoroacetic acid; HOBt or HOBT for hydroxybenzotriazole; DME for1,2-dimethoxyethane; and DMAP for N,N-dimethylaminopyridine.

Cap-1

Cap-1, Step a

2,6-Dimethyl-4H-pyran-4-one (15 g, 121 mmol) was dissolved in ethanol(300 mL) and 10% Pd/C (1.28 g, 1.21 mmol) was added. The mixture washydrogenated in a Parr shaker under H₂ (70 psi) at room temperature for72 hrs. The reaction mixture was filtered through a pad of diatomaceousearth (Celite®) and washed with ethanol. The filtrate was concentratedin vacuum and the residue was purified via flash chormatography (10% to30% EtOAc/Hex). Two fractions of clear oils were isolated. The firsteluting fractions were a mixture of(2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-ol (Cap-1, step a) and(2R,4s,6S)-2,6-dimethyltetrahydro-2H-pyran-4-ol (1.2 g) while the lattereluting fractions corresponded to only Cap-1, step a (10.73 g). ¹H NMR(500 MHz, CDCl₃) δ ppm 3.69-3.78 (1H, m), 3.36-3.47 (2H, m), 2.10 (1H,br. s.), 1.88 (2H, dd, J=12.05, 4.73 Hz), 1.19 (6H, d, J=6.10 Hz), 1.10(2H, q, J=10.70 Hz). ¹³C NMR (126 MHz, CDCl₃) δ ppm 71.44 (2C), 67.92(1C), 42.59 (2C), 21.71 (2C).

Cap-1, Step b

DEAD (166 mL, 330 mmol) was added drop wise to a solution of Cap-1, stepa (10.73, 82 mmol), 4-nitrobenzoic acid (48.2 g, 288 mmol) and Ph₃P (86g, 330 mmol) in benzene (750 mL). Heat evolution was detected and theresulting amber solution was stirred at ambient temperature for 18 h.Solvent was removed under reduced pressure and the residue wastriturated with Et₂O (200 mL) to remove triphenylphosphine oxide (10 g).The remaining mixture was purified via Biotage® (0 to 5% EtOAc/Hex; 300g column×4). A white solid corresponding to Cap-1, step b (19.36 g) wasisolated. ¹H NMR (500 MHz, CDCl₃) δ ppm 8.27-8.32 (2H, m), 8.20-8.24(2H, m), 5.45 (1H, quin, J=2.82 Hz), 3.92 (2H, dqd, J=11.90, 6.10, 1.53Hz), 1.91 (2H, dd, J=14.80, 2.29 Hz), 1.57 (2H, dt, J=14.65, 3.05 Hz),1.22 (6H, d, J=6.10 Hz). ¹³C NMR (126 MHz, CDCl₃) δ ppm 163.81 (1C),150.55 (1C), 135.94 (1C), 130.64 (2C), 123.58 (2C), 70.20 (1C), 68.45(2C), 36.95 (2C), 21.84 (2C). LC-MS: Anal. Calcd. for [M]⁺ C₁₄H₁₂NO₅:279.11. found 279.12.

Cap-1, Step c

A solution of LiOH (8.30 g, 347 mmol) in water (300 mL) was added to asolution of Cap-1, step b (19.36 g, 69.3 mmol) in THF (1000 mL) and theresulting mixture was stirred at ambient temperature for 16 h. THF wasremoved under reduced pressure and the aqueous layer was diluted withmore water (200 mL) and extracted with EtOAc (3×200 mL). The combinedorganic layers were dried (MgSO₄), filtered and concentrated undervacuum. An oily residue with a white solid was recovered. The mixturewas triturated with hexanes and the solid was removed by filtration toyield a clear oil corresponding to Cap-1, step c (8.03 g). ¹H NMR (500MHz, CDCl₃) δ ppm 4.21 (1H, quin, J=2.82 Hz), 3.87-3.95 (2H, m), 1.72(1H, br. s.), 1.63 (2H, dd, J=14.34, 2.14 Hz), 1.39-1.47 (2H, m), 1.17(6H, d, J=6.41 Hz). ¹³C NMR (126 MHz, CDCl₃) δ ppm 67.53 (2C), 64.71(1C), 39.99 (2C), 21.82 (2C).

Cap-1, Step d

p-Tosyl chloride (23.52 g, 123 mmol) was added to a solution of Cap-1,step c (8.03 g, 61.7 mmol) and pyridine (19.96 mL, 247 mmol) in CH₂Cl₂(750 mL) at room temperature and stirred for 36 h. As the reaction didnot proceed to completion, CH₂Cl₂ was removed under reduced pressure andstirring continued for another 48 h. The mixture was then added toCH₂Cl₂ (100 mL) and water (100 mL) and stirred at ambient temperaturefor 2 h. The mixture was separated and the organic layer was the washedthoroughly with 1N aq. HCl (2×50 mL). The organic layer was then dried(MgSO₄), filtered and concentrated. A yellow oil corresponding to Cap-1,step d (14.15 g) was isolated, which solidified under vacuum as anoff-white solid. ¹H NMR (500 MHz, CDCl₃) δ ppm 7.80 (2H, d, J=8.24 Hz),7.35 (2H, d, J=7.93 Hz), 4.88 (1H, quin, J=2.82 Hz), 3.79-3.87 (2H, m),2.46 (3H, s), 1.76 (2H, dd, J=14.50, 2.59 Hz), 1.36 (2H, ddd, J=14.34,11.60, 2.75 Hz), 1.12 (6H, d, J=6.10 Hz). ¹³C NMR (126 MHz, CDCl₃) δ ppm144.64 (1C), 134.24 (1C), 129.82 (2C), 127.61 (2C), 77.34 (1C), 67.68(2C), 37.45 (2C), 21.61 (1C), 21.57 (2C). LC-MS: Anal. Calcd. for[2M+H]⁺ C₂₈H₄₁O₈S₂: 569.22. found 569.3.

Cap-1, Step e

LiHMDS (29.7 mL, 29.7 mmol, 1 M in THF) was added to a solution ofCap-1, step d (7.05 g, 24.8 mmol) and benzyl2-(diphenylmethyleneamino)acetate (8.57 g, 26.0 mmol) in toluene (80 mL)at room temperature in a pressure tube and the resulting mixture wasthen stirred for 5 h at 100° C. The reaction was quenched with water(100 mL), extracted with EtOAc, washed with water, dried over MgSO₄,filtrated, and concentrated in vacuum. The residue was purified viaBiotage® (0% to 15% EtOAc/Hex; 240 g column) and a yellow oilcorresponding to Cap-1, step e (8.76 g) was isolated as a racemicmixture. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.62-7.71 (2H, m), 7.30-7.45(11H, m), 7.05 (2H, dd, J=7.65, 1.63 Hz), 5.13-5.22 (2 H, m), 3.89 (1H,d, J=6.78 Hz), 3.46 (2H, dquind, J=11.27, 5.90, 2.01 Hz), 2.34-2.45 (1H,m), 1.58-1.66 (1H, m), 1.34-1.43 (1H, m), 1.19 (3H, d, J=6.02 Hz),1.03-1.16 (4H, m), 0.83-0.97 (1H, m). ¹³C NMR (101 MHz, CDCl₃) δ ppm170.84 (1C), 170.68 (1C), 139.01 (1C), 135.96 (1C), 135.51 (1C), 130.04(1C), 128.49 (2C), 128.20 (1C), 128.09 (4C), 127.97 (2C), 127.85 (1C),127.67 (2C), 127.47 (2C), 72.76 (1C), 72.46 (1C), 69.77 (1C), 65.99(1C), 39.11 (1C), 35.90 (1C), 35.01 (1C), 21.74 (1C), 21.65 (1C). LC-MSAnal. Calcd. for [2M+Na]⁺ C₅₈H₆₂N₂NaO₆: 905.45. found 905.42.

Cap-1, Step f

Cap-1, Step e (8.76 g, 19.84 mmol) was dissolved in THF (100 mL) andtreated with 2 N HCl in water (49.6 mL, 99 mmol). The resulting clearsolution was stirred at ambient temperature for 4 h and then THF wasremoved under reduced pressure. The remaining aqueous layer wasextracted with EtOAc (3×30 mL) and concentrated under vacuum, to affordthe corresponding crude amine. The residue was taken up in CH₂Cl₂ (100mL) and charged with DIEA (11.8 mL, 67.6 mmol) and methyl chloroformate(1.962 mL, 25.3 mmol). The resulting solution was stirred at ambienttemperature for 2 h. The reaction mixture was diluted with CH₂Cl₂ (50mL) and washed with water (100 mL) and brine (100 mL). The organic layerwas dried (MgSO₄), filtered and concentrated. The residue was purifiedvia Biotage® (15% to 25% EtOAc/Hex; 80 g column) A clear colorless oilcorresponding to racemic Cap-1, step f (5.27 g) was recovered. ¹H NMR(400 MHz, CDCl₃) δ ppm 7.32-7.41 (5H, m), 5.13-5.28 (3H, m), 4.36 (1H,dd, J=8.16, 4.64 Hz), 3.69 (3H, s), 3.30-3.47 (2 H, m), 2.00-2.16 (1H,m), 1.52 (1H, d, J=12.55 Hz), 1.33 (1H, d, J=12.30 Hz), 1.15 (6H, dd,J=6.02, 5.02 Hz), 0.88-1.07 (2H, m). ¹³C NMR (101 MHz, CDCl₃) δ ppm171.39 (1C), 156.72 (1C), 135.20 (2C), 128.60 (2C), 128.57 (1C), 128.52(2C), 72.77 (1C), 72.74 (1C), 67.16 (1C), 57.81 (1C), 52.40 (1C), 38.85(1C), 35.56 (1C), 34.25 (1C), 21.94 (2C). LC-MS: Anal. Calcd. for [M+H]⁺C₁₈H₂₆NO₅: 336.18. found 336.3.

A chiral method was developed to separate the racemic mixture by using20% ethanol as the modifier on a CHIRALPAK® AS-H column (50×500 mm, 20μm) (Wavelength=220 nm, Flow rate=100 mL/min for 22 min, Solvent A=0.1%diethylamine in heptanes, Solvent B=EtOH). The two separated isomers,corresponded to (S)-benzyl2-((2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-((methoxycarbonyl)amino)acetate(Cap-1, step E 1) (Rt=9.8 min, 2.2 g) and (R)-benzyl2-((2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-((methoxycarbonyl)amino)acetate(Cap-1, step f.2) (Rt=16.4 min, 2.1 g) and they each exhibited the sameanalytical data as the corresponding mixture (see above).

Cap-1

(S)-benzyl2-((2R,4r,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-((methoxycarbonyl)amino)acetate(Cap-1, step £1) (2.2 g, 6.6 mmol) was dissolved in MeOH (50 mL) in aParr bottle and charged with 10% Pd/C (0.349 g, 0.328 mmol). Thesuspension was then placed in a Parr shaker and the mixture was flushedwith N₂ (3×), placed under 40 psi of H₂ and shaken at room temperaturefor 15 h. The catalyst was filtered off through a pad of diatomaceousearth (Celite®) and the solvent was removed under reduced pressure, toyield an amber solid corresponding to Cap-1 (1.6 g). ¹H NMR (500 MHz,DMSO-d₆) δ ppm 12.74 (1H, br. s.), 7.35 (1 H, d, J=6.10 Hz), 3.85 (1H,br. s.), 3.53 (3H, s), 3.35 (2H, ddd, J=15.95, 9.99, 6.10 Hz), 1.97 (1H,br. s.), 1.48 (2H, t, J=13.28 Hz), 1.06 (6H, d, J=6.10 Hz), 0.82-1.00(2H, m). ¹³C NMR (101 MHz, DMSO-d₆) δ ppm 176.93 (1C), 156.72 (1C,),72.10 (1C), 71.92 (1C), 58.54 (1C), 51.35 (1C), 36.88 (1C), 35.82 (1C),34.71 (1C), 21.90 (2C). Note: The absolute stereochemistry of Cap-1 wasdetermined by single crystal X-ray analysis of an ester analog preparedfrom Cap-1 and (S)-phenethanol.

Cap-2

(S)-2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(methoxycarbonylamino)acetic acid Cap-2, Step a

-   Reference: S. Danishefsky; et al. J. Org. Chem., 1982, 47, 1597.

Boron trifluoride etherate (3.81 mL, 30.5 mmol) was added dropwise to astirred and cooled (−78° C.) solution of(E)-(4-methoxybuta-1,3-dien-2-yloxy)trimethylsilane (5.0 g, 29 mmol) andacetaldehyde (3.28 mL, 58.0 mmol) in diethyl ether (100 mL) undernitrogen. The reaction was stirred at −78° C. for 2.5 h and thenquenched with sat. aq. NaHCO₃ (40 mL), allowed to warm to RT and stirredON. The layers were separated and the aqueous layer was extracted withdiethyl ether (2×50 mL). The combined organic layers were dried (MgSO₄),filtered and concentrated to a yellow/orange oil. The crude oil waspurified with a Biotage®Horizon (110 g SiO₂, 25-40% EtOAc/hexanes) toyield racemic 2-methyl-2H-pyran-4(3H)-one (Cap-2, step a) (2.2 g) as ayellow oil. ¹H NMR (400 MHz, CDCl₃-d) δ ppm 7.35 (d, J=6.0 Hz, 1H), 5.41(dd, J=6.0, 1.0 Hz, 1H), 4.51-4.62 (m, 1H), 2.41-2.57 (m, 2H), 1.47 (d,J=6.3 Hz, 3H).

Cap-2, Step b

-   Reference: Reddy, D. S.; et al. J. Org. Chem. 2004, 69, 1716-1719.

A solution of 1.6M methyllithium in diethyl ether (20.9 mL, 33.4 mmol)was added to a stirred slurry of copper(I) iodide (4.25 g, 22.30 mmol)in diethyl ether (30 mL) at 0° C. and under nitrogen. The reaction wasstirred at 0° C. for 20 min and then racemic 2-methyl-2H-pyran-4(3H)-one(1.25 g, 11.2 mmol) in diethyl ether (12.0 mL) was added over 10 min.The reaction was allowed to warm to RT and stirred 2 h. The reactionmixture was poured into sat NH₄Cl (aq) and stirred 20 min. The solutionwas extracted with diethyl ether (4×60 mL) and the combined organicswere washed with brine (˜80 mL), dried (MgSO₄), filtered andconcentrated to yield racemic(2R,6R)-2,6-dimethyldihydro-2H-pyran-4(3H)-one (Cap-2, step b) (1.34 g)as an orange oil. ¹H NMR (400 MHz, CDCl₃) δ ppm 4.28-4.39 (m, 2H), 2.55(ddd, J=14.1, 4.8, 1.5 Hz, 2H), 2.24 (ddd, J=14.1, 6.5, 1.5 Hz, 2H),1.28 (d, J=6.3 Hz, 6 H).

Cap-2, Step c

Sodium borohydride (0.354 g, 9.36 mmol) was added in portions to astirred solution of racemic(2R,6R)-2,6-dimethyldihydro-2H-pyran-4(3H)-one (Cap-2, step b) (1.2 g,9.4 mmol) in MeOH (30 mL) at 0° C. The solution was stirred 10 min at 0°C., warmed to RT and stirred 1 h. The reaction was poured into sat NH₄Cl(˜50 mL), stirred 20 min and then partially concentrated (to ˜½ volume).A precipitate formed, water was added until homogeneous and then thesolution was extracted with DCM (3×60 mL). The aqueous layer wasacidified with 1N HCl and then extracted with DCM (3×60 mL). Thecombined organics were dried with Na₂SO₄, filtered and concentrated toform a cloudy yellow oil (1.08 g). The crude oil was dissolved into DCM(8.0 mL) and then p-tosyl-Cl (2.68 g, 14.0 mmol) and pyridine (1.51 mL,18.7 mmol) were added and the reaction was allowed to stir at RT for2.5d. The reaction was diluted with sat NH₄Cl (˜60 mL) and extractedwith DCM (3×30 mL). The combined organic phase was dried (MgSO₄),filtered and concentrated to a brown oil. The oil was purified on aBiotage® Horizon (80 g SiO₂, 10-25% EtOAc/hexanes) to yield racemic(2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl-4-methylbenzenesulfonate(Cap-2, step c) (1.63 g) as a viscous clear colorless oil. LC-MSretention time 3.321 min; m/z 284.98 [M+H]⁺. LC data was recorded on aShimadzu LC-10AS liquid chromatograph equipped with a Phenomenex-Luna 3uC18 2.0×50 mm column using a SPD-10AV UV-Vis detector at a detector wavelength of 220 nM. The elution conditions employed a flow rate of 0.8mL/min, a gradient of 100% solvent A/0% solvent B to 0% solvent A/100%solvent B, a gradient time of 4 min, a hold time of 1 min, and ananalysis time of 5 min where solvent A was 5% MeOH/95% H₂O/10 mMammonium acetate and solvent B was 5% H₂O/95% MeOH/10 mM ammoniumacetate. MS data was determined using a Micromass Platform for LC inelectrospray mode. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.81 (d, J=8.3 Hz, 2H),7.36 (d, J=8.0 Hz, 2H), 4.81-4.92 (m, 1H), 4.17-4.26 (m, 1H), 3.78-3.87(m, 1H), 2.47 (s, 3H), 1.91-1.99 (m, 1H), 1.78-1.86 (m, 1H), 1.65-1.72(m, 1H), 1.46 (ddd, J=12.9, 9.4, 9.3 Hz, 1H), 1.20 (dd, J=6.5, 4.8 Hz,6H).

The racemic mixture was separated into the individual enantiomers inmultiple injections using chiral preparative SFC purification (ChiralpakAD-H preparative column, 30×250 mm, 5 μm, 10% 1:1 EtOH/heptane in CO₂,70 mL/min. for 10 min) to yield(2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl-4-methylbenzenesulfonate(Cap-2, step c.1) (577 mg) as the first eluting peak and(2S,6S)-2,6-dimethyltetrahydro-2H-pyran-4-yl-4-methylbenzenesulfonate(Cap-2, step c.2) (588 mg) as the second eluting peak. Each enantiomerwas isolated as a clear colorless oil which solidified to a white solidupon standing.

Cap-2, Step d

In a 48 mL pressure tube, (2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl4-methylbenzenesulfonate (Cap-2, step c.1) (575 mg, 2.02 mmol) andbenzyl 2-(diphenylmethyleneamino)acetate (733 mg, 2.22 mmol) werestirred in THF (2 mL) and toluene (10 mL). The clear colorless solutionwas flushed with nitrogen and then LiHMDS (1.0M in THF) (2.22 mL, 2.22mmol) was added and the vessel was sealed and heated at 100° C. for 8 h.The reaction was cooled to RT, poured into ½ sat NH₄Cl (aq) (˜50 mL) andextracted with EtOAc (3×30 mL). The combined organic layers were washedwith brine, dried (MgSO₄), filtered and concentrated to a crude orangeoil. The oil was purified on a Biotage® Horizon (40 g SiO₂, 10-25%EtOAc/hexanes) to yield impure desired product (501 mg) as an orangeoil. This material was repurified on a Biotage® Horizon (25 g SiO₂,6-12% EtOAc/hexanes) to yield an ˜1:1 mixture of diastereomers (Cap-2,step d) (306 mg) as a viscous orange oil.

The mixture was separated into the individual diastereomers in multipleinjections using chiral preparative SFC purification (Chiralcel OJ-Hpreparative column, 30×250 mm, 5 μm, 10% 1:1 EtOH/heptane in CO₂ @150bar, 70 mL/min. for 10 min) to yield (R)-benzyl2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(diphenylmethyleneamino)acetate(Cap-2, step d.1) (124 mg) as the first eluting peak and (S)-benzyl2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(diphenylmethyleneamino)acetate(Cap-2, step d.2) (129 mg) as the second eluting peak. Each diastereomerwas isolated as a viscous yellow oil.

Analytical data for (R)-benzyl2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(diphenylmethyleneamino)acetate(Cap-2, step d.1): ¹H NMR (400 MHz, D₄-MeOH) δ ppm 7.57-7.61 (m, 2H),7.41-7.48 (m, 4H), 7.33-7.40 (m, 7H), 7.03-7.08 (m, 2H), 5.22 (d, J=12.1Hz, 1H), 5.16 (d, J=12.1 Hz, 1H), 4.09-4.19 (m, 1 H), 3.84 (d, J=6.8 Hz,1H), 3.75-3.83 (m, 1H), 2.53-2.64 (m, 1H), 1.58-1.65 (m, 1H), 1.33-1.43(m, 1H), 1.26-1.32 (m, 1H), 1.24 (d, J=7.0 Hz, 3H), 1.10 (d, J=6.0 Hz,3H), 0.98-1.08 (m, 1H). LC-MS retention time 4.28 min; m/z 442.16[M+H]⁺. LC data was recorded on a Shimadzu LC-10AS liquid chromatographequipped with a Phenomenex-Luna 3u C18 2.0×50 mm column using a SPD-10AVUV-Vis detector at a detector wave length of 220 nM. The elutionconditions employed a flow rate of 0.8 mL/min, a gradient of 100%solvent A/0% solvent B to 0% solvent A/100% solvent B, a gradient timeof 4 min, a hold time of 1 min, and an analysis time of 5 min wheresolvent A was 5% MeOH/95% H₂O/10 mM ammonium acetate and solvent B was5% H₂O/95% MeOH/10 mM ammonium acetate. MS data was determined using aMicromass Platform for LC in electrospray mode.

Analytical data for (S)-benzyl2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(diphenylmethyleneamino)acetate(Cap-2, step d.2): ¹H NMR (400 MHz, D₄-MeOH) δ ppm 7.57-7.61 (m, 2H),7.41-7.50 (m, 4H), 7.33-7.40 (m, 7H), 7.04-7.08 (m, 2H), 5.22 (d, J=12.1Hz, 1H), 5.16 (d, J=12.1 Hz, 1H), 4.20 (qd, J=6.4, 6.3 Hz, 1H), 3.86 (d,J=6.5 Hz, 1H), 3.74-3.83 (m, 1H), 2.53-2.64 (m, 1H), 1.60 (td, J=12.7,5.6 Hz, 1H), 1.38-1.51 (m, 2H), 1.26 (d, J=7.0 Hz, 3H), 1.04 (d, J=6.0Hz, 3H), 0.79-0.89 (m, 1H). LC-MS retention time 4.27 min; m/z 442.17[M+H]⁺. LC data was recorded on a Shimadzu LC-10AS liquid chromatographequipped with a Phenomenex-Luna 3u C18 2.0×50 mm column using a SPD-10AVUV-Vis detector at a detector wave length of 220 nM. The elutionconditions employed a flow rate of 0.8 mL/min, a gradient of 100%solvent A/0% solvent B to 0% solvent A/100% solvent B, a gradient timeof 4 min, a hold time of 1 min, and an analysis time of 5 min wheresolvent A was 5% MeOH/95% H₂O/10 mM ammonium acetate and solvent B was5% H₂O/95% MeOH/10 mM ammonium acetate. MS data was determined using aMicromass Platform for LC in electrospray mode.

Cap-2, Step e

(S)-Benzyl2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(diphenylmethyleneamino)acetate(Cap-2, step d.2) (129.6 mg, 0.294 mmol) was dissolved in THF (2 mL) andthen treated with 2N HCl (1.0 mL, 2.1 mmol) in water. The reaction wasstirred for 2 h and then concentrated under a stream of nitrogenovernight. The crude residue was dissolved in DCM (2 mL) and DIPEA (0.21mL, 1.2 mmol) and then treated with methyl chloroformate (0.032 mL, 0.41mmol) and stirred at RT for 4 h. The reaction was diluted with water(˜2.5 mL) and extracted with DCM (4×2 mL). The combined organic phasewas concentrated under a stream of nitrogen overnight and the residuewas purified with a Biotage® Horizon (4 g SiO₂, 10-50% EtOAc/hexanes) toyield (S)-benzyl2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(methoxycarbonylamino)acetate(Cap-2, step e) (56 mg) as a colorless glass. LC-MS retention time 3.338min; m/z 335.99 [M+H]⁺. LC data was recorded on a Shimadzu LC-10ASliquid chromatograph equipped with a Phenomenex-Luna 3u C18 2.0×50 mmcolumn using a SPD-10AV UV-Vis detector at a detector wave length of 220nM. The elution conditions employed a flow rate of 0.8 mL/min, agradient of 100% solvent A/0% solvent B to 0% solvent A/100% solvent B,a gradient time of 4 min, a hold time of 1 min, and an analysis time of5 min where solvent A was 5% MeOH/95% H₂O/10 mM ammonium acetate andsolvent B was 5% H₂O/95% MeOH/10 mM ammonium acetate. MS data wasdetermined using a Micromass Platform for LC in electrospray mode. ¹HNMR (400 MHz, D₄-MeOH) δ ppm 7.29-7.42 (m, 5H), 5.28 (d, J=12.0 Hz, 1H),5.09 (d, J=12.0 Hz, 1H), 4.10-4.20 (m, 2H), 3.68-3.78 (m, 1H), 3.65 (s,3H), 2.22-2.36 (m, 1H), 1.42-1.54 (m, 2H), 1.29-1.38 (m, 1H), 1.17 (d,J=6.8 Hz, 3H), 1.04 (d, J=6.0 Hz, 3H), 0.89-1.00 (m, 1H).

Cap-2

(S)-Benzyl2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(methoxycarbonylamino)acetate(Cap-2, step e) (56 mg, 0.167 mmol) was dissolved in MeOH (4 mL) andthen treated with 10% Pd/C (12 mg, 0.012 mmol). The reaction mixture wasvacuum flushed with nitrogen (4×) and then with hydrogen (4×) andstirred under a balloon of hydrogen overnight. The reaction was filteredthrough Celite® and concentrated to yield(S)-2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(methoxycarbonylamino)aceticacid (Cap-2) (41 mg) as a colorless oil. ¹H NMR (400 MHz, D₄-MeOH) δ ppm4.22 (quin, J=6.4 Hz, 1H), 4.04-4.11 (m, 1H), 3.78-3.87 (m, 1H), 3.66(s, 3H), 2.26-2.39 (m, 1H), 1.63 (d, J=13.1 Hz, 1H), 1.51-1.60 (m, 1H),1.42-1.49 (m, 1H), 1.27 (d, J=7.0 Hz, 3H), 1.11 (d, J=6.3 Hz, 3H),0.97-1.08 (m, 1H).

Note: The absolute stereochemistry of Cap-2 was determined by singlecrystal X-ray analysis of an amide analog prepared from an epimer ofCap-2((R)-2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-((methoxycarbonyl)amino)aceticacid) and (S)-1-(naphthalen-2-yl)ethanamine

An alternative synthesis of Cap-2 is illustrated in Scheme-1. The firstfour steps resulting in (R)-2-methyl-2H-pyran-4(3H)-one were performedusing adaptations of a reported procedure (Anderson, K. R; et al. Org.Proc. Res. Dev. 2010, 14, 58). The conversion of(R)-2-methyl-2H-pyran-4(3H)-one to(2R,6R)-2,6-dimethyldihydro-2H-pyran-4(3H)-one was accomplished withadaptations of a reported procedure (Reddy, D. S.; et al. J. Org. Chem.2004, 69, 1716). Additional details are provided below.

(R)-Ethyl 3-(tert-butyldimethylsilyloxy)butanoate

tert-Butylchlorodimethylsilane (547 g, 3.63 mol) was added to a stirredsolution of (R)-ethyl 3-hydroxybutanoate (400 g, 3.03 mol) in DCM (800mL) under nitrogen at 0° C. Imidazole (412 g, 6.05 mol) was addedportion wise over 20 min to the reaction mixture during which time themixture became a thick white slurry. Additional DCM (175 mL) was addedand the mixture was allowed to warm to room temperature and stirred for16 h. The white solid was removed by filtration, rinsed with DCM (500mL), partitioned between water (1 L) and DCM (500 mL), and the aqueouslayer was further extracted with additional DCM (500 mL). The filtratewas combined with the organic layers and washed with water (500 mL) andbrine, dried over MgSO₄ and filtered. The product was concentrated anddried under high vacuum to yield (R)-ethyl3-(tert-butyldimethylsilyloxy)butanoate (777.5 g), containingunidentified impurities. The analytical data of the crude productcomplied with data reported in the literature. The material was usedwithout additional purification.

(R)-3-(tert-Butyldimethylsilyloxy)-N-methoxy-N-methylbutanamide

A solution of 2M isopropylmagnesium chloride in THF (800 mL, 1.60 mol)was added dropwise via cannula over 60 min to a stirred solution of(R)-ethyl 3-(tert-butyldimethylsilyloxy)butanoate (131 g, 532 mmol) andN,O-dimethylhydroxylamine/HCl (80 g, 824 mmol) in dry THF (850 mL) whilemaintaining the internal temperature between −30° C. and −20° C. Thesuspension was allowed to stir for 3 h between −20 and −10° C., andquenched while cold with saturated aqueous ammonium chloride solution(400 mL). The reaction mixture was partitioned between water (200 mL)and diethyl ether (500 mL) and the aqueous phase was further extractedwith diethyl ether (1 L). The combined organic phases were washed withbrine, dried over MgSO₄, filtered, concentrated and dried under highvacuum to yield(R)-3-(tert-butyldimethylsilyloxy)-N-methoxy-N-methylbutanamide. Thereaction was repeated 6 times to yield a total of 717 g material. Theanalytical data complied with data reported in the literature. Thematerial was used without additional purification.

N-Ethylidenepiperidin-1-amine

-   For relevant references, see: (a) Marques-Lopez, E.; et al. Eur. J.    Org. Chem. 2008, 20, 3457. (b) Corey, E.; et al. Chem. Ber. 1978,    111, 1337. (c) Chudek, J. A. et al. J. Chem. Soc. Perkin Trans 2    1985, 8, 1285.

Piperidin-1-amine (754 mL, 6.99 mol) was added dropwise over 60 min toacetaldehyde maintained at a 0° C. (304 mL, 5.38 mol) while stirringunder nitrogen. [Caution: The addition is exothermic. On this scale, thedropping funnel containing piperidin-1-amine became hot due toacetaldehyde that had vaporized and condensed in the dropping funnel].After 1 h at 0° C. and 1 h at rt, the reaction flask was equipped with a16-in reflux condenser and was warmed to 40° C. and stirred for 20 h.The reaction mixture was cooled to RT and partitioned between diethylether (700 mL) and brine (300 mL). The organic layer was washed withwater and the aqueous layer was extracted with diethyl ether (2×500 mL).The combined organic layers were washed with brine, dried over MgSO₄,filtered, concentrated and dried under high vacuum for 8 h to afford(E)-N-ethylidenepiperidin-1-amine (772 g) (contained impurities) as ayellow oil. The analytical data complied with data reported in theliterature. The material was used without additional purification.

(E)-5-((R)-tert-Butyldimethylsilyloxy)-1-(piperidin-1-ylimino)hexan-3-one

n-BuLi in hexanes (2.5 M, 644 mL, 1.61 mol) was added over 40 min to astirred solution of diisopropylamine (245 mL, 1.72 mol) in dry THF (2.3L) at 0° C. under nitrogen. The mixture was stirred at 0° C. for 30 minand then a solution of (E)-N-ethylidenepiperidin-1-amine (203 g, 1.61mol) in dry THF (100 mL) was added dropwise. The reaction mixture wasstirred at 0° C. for 2 h and the suspension cooled to −78° C. andtreated dropwise with a solution of(R)-3-(tert-butyldimethylsilyloxy)-N-methoxy-N-methylbutanamide (280.4g, 1.073 mol) in dry THF (100 mL). The reaction mixture was stirred for3 h at −78° C., allowed to slowly warm to room temperature and thenstirred for 16 h. The reaction mixture was partitioned between water(600 mL) and diethyl ether (1.5 L), and the aqueous layer was furtherextracted with diethyl ether. The combined organic layers were washedwith brine, dried over MgSO₄, filtered and concentrated to yield anamber-colored oil. The crude oil was purified through a short column (3L silica gel in a 4 L sintered glass funnel was pre-equilibrated with 5%EtOAc/hexanes, then the crude oil was dissolved in 50 mL of DCM/hexanes(1:5) and loaded onto the top of the silica gel, and, finally, thecolumn was eluted with 5-40% EtOAc/hexanes) to yield(E)-5-((R)-tert-butyldimethylsilyloxy)-1-(piperidin-1-ylimino)hexan-3-one.Impure fractions were combined, concentrated and repurified with aBiotage® Horizon (300 g SiO₂, 15-45% EtOAc/hexanes) to yield additionalproduct (230 g total). The analytical data complied with data reportedin the literature. The material was used without additionalpurification.

(R)-2-Methyl-2H-pyran-4(3H)-one

Amberlyst-15 (122 g, 372 mmol) (dry form, pale brown beads, Alfa Aesar,Stock #89079 or L14146) was added in one portion to a stirred solutionof(E)-5-((R)-tert-butyldimethylsilyloxy)-1-(piperidin-1-ylimino)hexan-3-one(121 g, 372 mmol) in dry THF (1.2 L). The mixture was refluxed for 2.5h, cooled, filtered, and concentrated to an amber-colored liquid. Thecrude liquid was purified through a short column (the crude product wasdissolved in 30 mL of 5% EtOAc/hexanes and loaded onto the top of 1.5 Lof silica gel in a 4 L sintered glass funnel that was pre-equilibratedwith 5% EtOAc/hex, and the column was eluted with 5-50% EtOAc/hexanes)to yield enone (R)-2-methyl-2H-pyran-4(3H)-one. Note: The solvent wasremoved by careful rotary evaporation (bath temperature was less than orequal to ambient temperature) but some desired product was detected inthe recovery trap. This material was concentrated to remove solventusing a 20-inch Vigreux column with a distillation head and combinedwith the original material. The total desired product was furtherpurified by distillation to afford a colorless oil (17.8 g) with boilingpoint of 68-70° C. at 10 mm Hg. Optical Rotation: +212.8 at c=0.46 g/100mL CHCl₃.

(2R,6R)-2,6-Dimethyldihydro-2H-pyran-4(3H)-one

Methyllithium in diethyl ether (1.6M, 218 mL, 349 mmol) was added in ˜10mL portions over 20 min to a stirred slurry of copper(I) iodide (44.3 g,233 mmol) in diethyl ether (350 mL) cooled to 0° C. under nitrogen. Thereaction mixture was stirred at 0° C. for 30 min and then(R)-2-methyl-2H-pyran-4(3H)-one (13.5 g, 116 mmol) in diethyl ether (150mL) was added dropwise over 30 min. The cold bath was removed and thereaction mixture was allowed to warm to rt and stirred for 3 h. Thereaction mixture was slowly added (over ˜5 min) to a stirred solution ofsat. NH₄Cl (aq) (˜750 mL) and ice. Additional diethyl ether (˜500 mL)and water (˜150 mL) were added and the solution, which contained a greyprecipitate, was stirred overnight. The layers were separated and theaqueous layer was extracted with diethyl ether (500 mL) and DCM (500mL). The combined organic phase was dried (MgSO₄), filtered andconcentrated (the rotary evaporator bath temperature was held at orbelow ambient temperature) to an orange oil. The crude oil was purifiedwith a Biotage® Horizon (240 g SiO₂, 1-4% diethyl ether in DCM, columnwas pre-equilibrated with DCM) to yield(2R,6R)-2,6-dimethyldihydro-2H-pyran-4(3H)-one (13.4 g, 86% pure by¹H-NMR) as a light yellow oil. [Note: The material was carefullyconcentrated and contained 10% w/w DCM by ¹H-NMR. ¹H-NMR analysis alsoindicated that the material was contaminated with ˜2.5% TBDMSOH carriedover from a previous reaction and 1.6% of(2R,6S)-2,6-dimethyldihydro-2H-pyran-4(3H)-one]. The analytical data ofthe product complied with the data reported in the literature, and thematerial was used without additional purification. ¹H-NMR (400 MHz,CDCl₃) δ 4.41-4.25 (m, 2H), 2.55 (ddd, J=14.1, 4.8, 1.4 Hz, 2H), 2.24(ddd, J=14.1, 6.5, 1.5 Hz, 2H), 1.28 (d, J=6.5 Hz, 6H).

Ethyl2-((2R,6R)-2,6-dimethyl-2H-pyran-4(3H,5H,6H)-ylidene)-2-formamidoacetate

Cuprous oxide (2.21 g, 15.4 mmol) was added to a stirred solution ofethyl 2-isocyanoacetate (12.7 mL, 116 mmol) in diethyl ether (300 mL)and the slurry was vigorously stirred for 10 min.(2R,6R)-2,6-Dimethyldihydro-2H-pyran-4(3H)-one (15.2 g, 103 mmol, 87%purity with 13% w/w DCM) in diethyl ether (100 mL) was added over 15 minand the reaction mixture was stirred at RT for 2 h. The reaction mixturewas cooled to 0° C. and treated with 1M KO-tBu (116 mL, 116 mmol) in THFadded in 10 mL portions over ˜15 min and then stirred at 0° C. for 1 h.A solution of acetic acid (6.66 mL, 116 mmol) in DCM (60 mL) was addedand the reaction mixture was allowed to warm to RT and stirred for 2 h.The crude reaction mixture was partitioned between water (˜300 mL) anddiethyl ether (˜300 mL) and the aqueous layer was further extracted withDCM (300 mL). The combined organic phase was dried (MgSO₄), filtered andconcentrated to a dark red oil, which solidified under vacuum. The crudematerial was purified with a Biotage® Horizon chromatography (300 gSiO₂; loaded onto column using a minimal amount of DCM; eluted with40-60% EtOAc/hexanes, holding at 40% for four column volumes). The earlyfractions of desired product also contained the cis-stereoisomer versionof the target product as an impurity. These impure fractions werecombined, concentrated to give 8.9 g of a light yellow oil that wasrepurified with a Biotage® Horizon chromatography (240 g silica gel;eluted with 40-65% EtOAc/hexanes, holding at 40% for five columnvolumes). All fractions with clean desired product from bothpurifications, as determined by TLC, were combined and concentrated toyield ethyl2-((2R,6R)-2,6-dimethyl-2H-pyran-4(3H,5H,6H)-ylidene)-2-formamidoacetate(18.8 g) as a light yellow solid contaminated with <1% (HPLC) of thecis-dimethyl isomeric variant.

The ¹H- and ¹³C-NMR data for the pure material presents as a 2:1 mixtureof amide rotamers: ¹H-NMR (400 MHz, CDCl₃) δ 8.24 (d, J=1.3 Hz, 0.66H),7.98 (d, J=11.5 Hz, 0.33H), 6.71 (br. s., 0.66H), 6.56 (d, J=12.0 Hz,0.33H), 4.32-4.00 (m, 4H), 3.05 (dd, J=14.3, 4.3 Hz, 0.33H), 2.92 (d,J=5.3 Hz, 1.33H), 2.82 (dd, J=14.3, 6.5 Hz, 0.33H), 2.62 (dd, J=13.9,3.9 Hz, 0.33H), 2.45 (dd, J=14.1, 3.8 Hz, 0.66H), 2.24 (dd, J=13.8, 7.3Hz, 0.33H), 2.14-2.04 (m, 0.66H), 1.37-1.29 (m, 3H), 1.28-1.18 (m, 6H).¹³C-NMR (101 MHz, CDCl₃) δ 164.5 (0.66C), 163.9 (0.33C), 163.8 (0.33C),159.3 (0.66C), 147.5 (0.33C), 147.0 (0.66C), 120.3 (0.33C), 118.6(0.66C), 68.1 (0.66C), 67.8 (0.33C), 67.4 (0.33C), 66.8 (0.66C), 61.1(0.33C), 60.9 (0.66C), 38.2 (0.66C), 37.4 (0.33C), 36.2 (0.33C), 36.0(0.66C), 20.5 (0.66C), 20.1 (0.33C), 19.5 (0.33C), 19.2 (0.66C), 13.8(s, 1C). LC-MS retention time 1.85 min; m/z 242.45 (M+H)⁺. LC data wasrecorded on a Shimadzu LC-10AS liquid chromatograph equipped with aPhenomenex-Luna 3μ C18 2.0×50 mm column using a SPD-10AV UV-Vis detectorat a detector wave length of 220 nM. The elution conditions employed aflow rate of 0.8 mL/min, a gradient of 100% solvent A/0% solvent B to 0%solvent A/100% solvent B, a gradient time of 4 min, a hold time of 1min, and an analysis time of 5 min. Solvent A was 10% acetonitrile/90%H₂O/0.1% trifluoroacetic acid, and solvent B was 10% H₂O/90%acetonitrile/0.1% trifluoroacetic acid. MS data was determined using aMicromass Platform for LC in electrospray mode.

(S)-Ethyl2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-formamidoacetate

Nitrogen was bubbled through a solution of ethyl2-((2R,6R)-2,6-dimethyl-2H-pyran-4(3H,5H,6H)-ylidene)-2-formamidoacetate(17.0 g, 70.5 mmol) in MeOH (480 mL) for 10 min in a 2.5 L Parrhydrogenation vessel. Then(−)-1,2-bis((2S,5S)-2,5-dimethylphospholano)ethane(cyclooctadiene)-rhodium(I) tetrafluoroborate (0.706 g, 1.27 mmol) was added and the reactionvessel was sealed, vacuum flushed with nitrogen (4×) and then vacuumflushed with hydrogen (4×). The reaction solution was shaken under 60psi of hydrogen for 3 d, removed from the shaker and concentrated to adark red oil. The crude oil was purified with a Biotage® Horizonchromatography (300 g SiO₂, 50-75% EtOAc/hexanes) to yield (S)-ethyl2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-formamidoacetate(17.4 g, contains 5.9% w/w solvents (EtOAc and DCM) by ¹H-NMR) as alight yellow viscous oil. The ¹H-NMR data indicates a 10:1 mixture oftwo amide rotamers. ¹H-NMR (400 MHz, CDCl₃) δ 8.26 (s, 0.9H), 8.01 (d,J=11.8 Hz, 0.1H), 6.15 (d, J=8.5 Hz, 0.9H), 5.96-5.84 (m, 0.1H), 4.68(dd, J=9.0, 5.0 Hz, 0.9H), 4.33-4.17 (m, 3H), 3.87 (dd, J=10.2, 6.4 Hz,0.1H), 3.75 (dqd, J=11.5, 6.0, 2.0 Hz, 1H), 2.41-2.29 (m, 0.9H),2.29-2.19 (m, 0.1H), 1.72-1.28 (m, 3H), 1.28-1.23 (m, 6H), 1.17-1.10 (m,3H), 1.09-0.96 (m, 1H). ¹³C-NMR (101 MHz, CDCl₃) δ 170.6, 160.3, 68.1,64.0, 61.4, 54.2, 36.0, 33.1, 30.6, 21.9, 16.7, 13.9. LC-MS retentiontime 1.64 min; m/z 244.25 (M+H)⁺. LC data were recorded on a ShimadzuLC-10AS liquid chromatograph equipped with a Phenomenex-Luna 3μ C182.0×50 mm column using a SPD-10AV UV-Vis detector at a detector wavelength of 220 nM. The elution conditions employed a flow rate of 0.8mL/min, a gradient of 100% solvent A/0% solvent B to 0% solvent A/100%solvent B, a gradient time of 4 min, a hold time of 1 min, and ananalysis time of 5 min. Solvent A was 10% acetonitrile/90% H₂O/0.1%trifluoroacetic acid, and solvent B was 10% H₂O/90% acetonitrile/0.1%trifluoroacetic acid. MS data were determined using a Micromass Platformfor LC in electrospray mode.

(S)-Ethyl2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(methoxycarbonyl-amino)acetate

In a 1 L flask equipped with a condenser which was stoppered with aseptum and vented with a needle, a solution of (S)-ethyl2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-formamidoacetate(17.6 g, 72.3 mmol) in ethanol (409 mL) and 1.5 N HCl(aq) (409 mL) washeated in a pre-equilibrated oil bath at 52° C. for 7.5 h. The volatilecomponent was removed under vacuum and the residue was azeotroped withEtOH (2×50 mL) and dried under vacuum overnight to afford a white foam.The white foam was dissolved into DCM (409 mL) andN,N-diisopropylethylamine (38.0 mL, 218 mmol) was added dropwise over 10min followed by methyl chlorocarbonate (8.40 mL, 109 mmol), also addeddropwise over 10 min. The reaction mixture was stirred at ambienttemperature for 4.5 h and then quenched carefully with 1 N HCl (100 mL).The layers were separated and the organic layer was washed with 1 N HCl(50 mL) and then with an aqueous basic solution (50 mL water+2 mL sat.NaHCO₃). The organic layer was dried (MgSO₄), filtered, andconcentrated. The residue was purified with a Biotage® (300 g SiO₂;sample was loaded onto column with a minimal amount of chloroform, andeluted with 30% EtOAc/hexanes,) to yield (S)-ethyl2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(methoxycarbonyl-amino)acetate(13.8 g) as a viscous yellow oil. ¹H-NMR (400 MHz, CDCl₃) δ 5.23 (d,J=8.8 Hz, 1H), 4.34-4.15 (m, 4H), 3.81-3.72 (m, 1H), 3.70 (s, 3H),2.35-2.23 (m, 1H), 1.63-1.51 (m, 2H), 1.34-1.27 (‘m’ & ‘t’ overlapped,J=7.0 Hz, 1H & 3H), 1.25 (d, J=6.8 Hz, 3H), 1.14 (d, J=6.0 Hz, 3H),1.11-0.99 (m, 1H). LC-MS retention time 2.89 min; m/z 296.23 (M+Na)⁺. LCdata were recorded on a Shimadzu LC-10AS liquid chromatograph equippedwith a Phenomenex-Luna 3μ C18 2.0×50 mm column using a SPD-10AV UV-Visdetector at a detector wave length of 220 nM. The elution conditionsemployed a flow rate of 0.8 mL/min, a gradient of 100% solvent A/0%solvent B to 0% solvent A/100% solvent B, a gradient time of 4 min, ahold time of 1 min, and an analysis time of 5 min. Solvent A was 10%MeOH/90% H₂O/0.1% trifluoroacetic acid, and solvent B was 10% H₂O/90%MeOH/0.1% trifluoroacetic acid. MS data were determined using aMicromass Platform for LC in electrospray mode.

Additional note: the above product was further purified by SFCchromatography (180 mg/injection, with a throughput of 7.2 g/hr) priorto the hydrolysis step to remove an unidentified impurity. Conditions:

Column: ChiralPak AD-H 25×3 cm, 5μ Column Temperature: 25° C.

Flow rate: 150 mL/min

Mobile Phase: CO₂/MeOH=93/7

Injection Volume: 1.0 mL (180 mg/mL)Injection Model: Stacked (injection/run time (min)=1.47/3.0)

Detector Wavelength: 220 nm

Sample Solvent: CH₃OH/CH₃CN=1:1(v/v)

(S)-2-((2R,6R)-2,6-Dimethyltetrahydro-2H-pyran-4-yl)-2-(methoxycarbonylamino)acetic acid (Cap-2)

Lithium hydroxide hydrate (8.51 g, 203 mmol) in H₂O (270 mL) was addeddropwise to a stirred solution of (S)-ethyl2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(methoxycarbonyl-amino)acetate(27.7 g, 101 mmol) in THF (405 mL) over 65 min, while maintaining aninternal temperature <5° C. The reaction mixture was stirred in an icebath for 40 min and then in a water bath at RT for 5 h. The reactionmixture was partially concentrated to remove the THF and thenpartitioned between water (250 mL) and DCM (250 mL). The aqueous layerwas adjusted to pH=2 with 1 N HCl (205 mL), and then extracted withisopropyl acetate (7×250 mL). The combined extracts were washed withbrine (1×600 mL), dried (MgSO₄), filtered and concentrated. Theresulting viscous oil was treated with toluene (100 mL) and ether (75mL), evaporated and dried in vacuo to afford Cap-2 (25.8 g, with 0.33eq. of toluene) as a white foam. Chiral SFC purity >99.9% (ChiralpakAD-H (25×0.46 cm, 5 μm) 5% EtOH in CO₂, 3 mL/min, 35° C., 220 nm, 100bar). HPLC purity 95.6% (Column: Sunfire C8 75×4.6 mm ID; 2.5 μm; 40°C.; Mobile Phase: A=Water w/ 0.1% Formic Acid; B═CH₃CN w/ 0.1% FormicAcid). LC/MS (ES+) 228.1 (M+H−H₂O)⁺. By ¹H-NMR the material containedabout 0.33 eq. of toluene. ¹H-NMR (400 MHz, CDCl₃) δ 5.29 (d, J=8.6 Hz,1H), 4.46-4.25 (m, 2H), 3.82 (dd, J=9.7, 5.9 Hz, 1H), 3.74 (s, 3H),2.46-2.30 (m, 1H), 1.76-1.55 (m, 2H), 1.51-1.35 (m, 1H), 1.29 (d, J=6.8Hz, 3H), 1.18 (d, J=6.2 Hz, 3H), 1.18-1.06 (m, 1H). ¹³C-NMR (101 MHz,CDCl₃) δ 174.1, 156.7, 68.3, 64.4, 57.5, 52.2, 35.8, 32.6, 30.3, 21.6,16.7.

EXAMPLE 1

Dimethyl(4,4′-biphenyldiylbis(1H-imidazole-4,2-diyl((2S,5S)-5-methyl-2,1-pyrrolidinediyl)((1S)-1-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-oxo-2,1-ethanediyl))biscarbamate

Example 1, Step a

The above compound was synthesized according to a literature protocol(J. Med. Chem., 2006, 49, 3520) with the following purificationmodifications: the crude material was recrystallized from EtOAc/hexanesat ambient temperature to afford Example 1, Step a, as a white crystal.¹H NMR (400 MHz, CDCl₃) δ ppm 4.32 (br m, 1H), 3.89 (br m, 1H), 2.40 (brm, 1H), 2.00 (m, 2H), 1.65 (m, 1H), 1.45 (s, 9H), 1.20 (d, J=5.6, 3H).LC/MS: Anal. Calcd. for [M+Na]⁺ C₁₁H₂₀NO₄Na: 252.12. found 252.21.

Example 1, Step b

To a mixture of Example 1, step a (7.12 g, 31.1 mmol) and1,1′-(biphenyl-4,4′-diyl)bis(2-bromoethanone) (6.0 g, 15 mmol) inacetonitrile (100 mL), i-Pr₂EtN (5.56 mL, 31.8 mmol) was added dropwise,and the reaction was stirred at room temperature for 3 hr. The volatilecomponent was removed in vacuo and the residue was partitioned betweenCH₂Cl₂ (100 mL) and sat. aq. NaHCO₃ (100 mL). The organic layer wasseparated and washed with water and brine, dried (Na₂SO₄), filtered, andconcentrated in vacuo to afford Example 1, step b as white foam (9.83g), which was used in the next step without purification. ¹H NMR (500MHz, CDCl₃) δ ppm 8.01 (4H, t, J=7.32 Hz), 7.73 (4H, d, J=6.71 Hz),5.18-5.66 (4H, m), 4.51 (1H, t, J=7.17 Hz), 4.42 (1H, t, J=7.32 Hz),4.06 (1H, d, J=3.36 Hz), 3.95 (1H, d, J=5.19 Hz), 2.27-2.37 (4H, m),2.01-2.17 (2H, m), 1.67-1.82 (2H, m), 1.39-1.52 (18H, m), 1.32 (6H, t,J=6.56 Hz). LC-MS: Anal. Calcd. for [M+H]⁺ C₃₈H₄₉N₂O₁₀ 693.34. found693.34.

Example 1, Step c

A mixture of Example 1, step b (9.83 g, 14.19 mmol) and ammonium acetate(10.94 g, 142 mmol) in xylene (160 mL) was heated in a pressure tube at140° C. for 4 hours. The volatile component was removed in vacuo and theresidue was partitioned between CH₂Cl₂ (140 mL) and water (100 mL). Theorganic layer was washed with saturated NaHCO₃ (100 mL), dried (Na₂SO₄)and concentrated in vacuo. The resultant crude material was purified viaBiotage® (20% to 50% EtOAc/Hex; 300 g column) to afford Example 1, stepc as light brown solid (4.3 g). ¹H NMR (500 MHz, DMSO-d₆) δ ppm 11.73(2H, br. s.), 7.83 (3H, d, J=7.63 Hz), 7.62-7.76 (5 H, m), 7.27-7.55(2H, m), 4.82 (2H, br. s.), 3.89 (2H, br. s.), 2.10 (6H, d, J=15.26 Hz),1.58-1.89 (2H, m), 1.11-1.52 (24H, m). LC/MS: Anal. Calcd. for [M+H]⁺C₃₈H₄₉N₆O₄: 653.37. found 653.60.

Alternative procedure: a mixture of Example 1, step b (157 g, 227 mmol),ammonium acetate (332 g, 4310 mmol), and imidazole (54.1 g, 795 mmol) intoluene (1.2 L) was heated at 80° C. for 1 h while sweeping the headspace with nitrogen. The temperature was increased to 85° C. and thereaction mixture stirred for 18 h. The solvent was removed and theresidue dissolved in DCM (2 L), washed with water (1 L) and sat. NaHCO₃(3 L) and then dried (MgSO₄), filtered and concentrated. The residue wasdissolved in MeOH (4 L), concentrated to −900 mL and allowed to stand atrt. After 2 h, the precipitated solid was collected by filtration,washed with MeOH and dried to yield a white solid. This material wasrecrystallized from methanol (dissolve in 4 L, then reduced to −0.9 L)and dried to afford Example 1, step c as a white solid (99.5 g). Thecrude compound from the mother liquor was purified by silica gelchromatography (300 g SiO₂, 20-50% EtOAc/hexanes) to yield additionalproduct as a white solid (13.0 g).

Example 1, Step d

4 N HCl in dioxane (8.23 mL, 32.9 mmol) was added dropwise to a CH₂Cl₂(100 mL) solution of Example 1, Step c (4.3 g, 6.6 mmol), and thereaction mixture was stirred at room temperature for 3 hours. Removal ofthe volatile component in vacuo afforded the HCl salt of Example 1, stepd as a yellow solid (3.6 g). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.55 (2H,br. s.), 9.73 (2H, br. s.), 8.12-8.26 (2H, m), 8.03 (4H, d, J=8.03 Hz),7.92 (4H, d, J=6.02 Hz), 5.07 (2H, m, J=7.53 Hz), 3.82 (2H, br. s.),2.53-2.65 (4H, m), 2.27 (2H, dd, J=12.42, 6.40 Hz), 1.85-1.99 (2H, m),1.45 (6H, d, J=6.27 Hz). LC/MS: Anal. Calcd. for [M+H]⁺ C₂₈H₃₃N₆:453.28. found 453.21.

The purity level of the above deprotected product could be enhanced byapplying the following recrystallization procedure: 2-propanol (242 mL)was added through an addition funnel over 15 min to a solution ofExample 1, step d (60.5 g, HPLC purity of 98.9%) in water (121 mL)stirring at 60° C., while maintaining an internal temperature between50° C. and 60° C. The solution was stirred for an additional 5 min, theheating mantle was removed and the mixture stirred at ambienttemperature overnight. The precipitated solid was collected byfiltration, washed with 2-propanol and dried under house vacuumovernight to give a yellow solid (54.4 g) with HPLC purity of 99.7%(Column: BEH C18 150 mm (L)×2.1 mm (ID), 1.7 μm, Mobile Phase: A: 0.05%TFA in water; B: 0.05% TFA in ACN). KF 15.3 wt % (6.0 mol of H₂O).

EXAMPLE 1

HATU (1.60 g, 4.21 mmol) was added to a solution of an HCl salt ofExample 1, step d (1.2 g, 2.0 mmol), Cap-1 (1.01 g, 4.11 mmol) and DIEA(2.1 mL, 12 mmol) in DMF (20 mL) and the resulting yellow solution wasstirred at RT for 3 h. The mixture was diluted with EtOAc (75 mL) andwashed with water (100 mL). The aqueous layer was back extracted withEtOAc (75 mL) and the combined organic layers were washed with 50%solution of sat. aqueous NaHCO₃ (100 mL), water (100 mL) and brine (100mL). It was then dried (MgSO₄), filtered and concentrated. The remainingresidue was diluted with CH₃OH and submitted to a reverse phase HPLCpurification: Solvent A: 5% MeCN/95% water/10 nM NH₄OAc; Solvent B: 95%MeCN/5% water/10 nM NH₄OAc; Column: Sunfire Prep C18 50×300 mm 10 u;Wavelength: 220 nM; Flow rate: 150 ml/min; Gradient: 0% B to 70% B over25 min with a 5 min hold time. Concentration under vacuum, yieldeddimethyl(4,4′-biphenyldiylbis(1H-imidazole-4,2-diyl((2S,5S)-5-methyl-2,1-pyrrolidinediyl)((1S)-1-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-oxo-2,1-ethanediyl)))biscarbamate(Example 1) as an off-white foam (1.2 g). Crystallization fromEtOAc/Petroleoum ether gave an amorphous off-white solid. ¹H NMR (500MHz, DMSO-d₆) (of the TFA salt) δ 7.92 (br. s., 8H), 7.59 (d, J=7.9 Hz,2H), 5.03 (t, J=8.5 Hz, 2H), 4.69-4.60 (m, 2H), 4.00 (t, J=8.4 Hz, 2H),3.55 (s, 6H), 3.36 (br. s., 1H), 3.33-3.26 (m, 2H), 3.21 (br. s., 2H),2.37 (br. s., 1H), 2.26 (d, J=11.3 Hz, 1H), 1.95-1.81 (m, 3H), 1.65 (d,J=10.7 Hz, 2H), 1.48 (d, J=6.4 Hz, 6H), 1.28-1.23 (m, 4H), 1.10-0.99 (m,16H), 0.90-0.75 (m, 4H)

LC/MS: Anal. Calcd. for [M+H]⁺ C₅₀H₆₇N₈O₈: 907.51. found 907.8. Rt=3.99min & >95% homogeneity index under the following LC condition—Column:Phenomenex-Luna 2.0×50 mm 3 um; Start % B=0; Final % B=100; GradientTime=10 min; Flow Rate=4 mL/Min; Wavelength=220; Solvent A=H₂O:ACN95%:5% 10 mm Ammonium Acetate; Solvent B═H₂O:ACN 5%:95% 10 mm AmmoniumAcetate.

EXAMPLE 2

Dimethyl(4,4′-biphenyldiylbis(1H-imidazole-4,2-diyl((2S,5S)-5-methyl-2,1-pyrrolidinediyl)((1S)-1-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-oxo-2,1-ethanediyl)))biscarbamate

HATU (63.6 mg, 0.167 mmol) was added to a stirred solution of(S)-2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(methoxycarbonylamino)aceticacid (Cap-2) (41 mg, 0.17 mmol) and the HCl salt of Example 1, step d(45.5 mg, 0.076 mmol) in DMF (0.9 mL) and DIPEA (0.11 mL, 0.61 mmol).The reaction mixture was stirred at RT for 4 h and then concentratedunder a stream of nitrogen ON. The residue was dissolved in MeOH,filtered and purified by prep HPLC (Phenomenex Luna C18(2) 100×30 mm, 10micron; MeOH/water w/ TFA buffer) to afford the TFA salt of dimethyl(4,4′-biphenyldiylbis(1H-imidazole-4,2-diyl((2S,5S)-5-methyl-2,1-pyrrolidinediyl)((1S)-1-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-oxo-2,1-ethanediyl)))biscarbamate(Example 2) (62.5 mg) as a light yellow solid. ¹H NMR indicates thepresence of a mixture of rotamers; for the major rotamer: ¹H NMR (400MHz, MeOH-d₄) δ 8.02-7.93 (m, 3H), 7.88 (br s, 7H), 5.18 (dd, J=10.7,7.2 Hz, 2H), 4.77 (quin, J=6.6 Hz, 2H), 4.27-4.17 (m, 2H), 4.15 (d,J=9.3 Hz, 2H), 3.72-3.66 (m, 2H), 3.67 (s, 6H), 2.72-2.50 (m, 2H),2.48-2.16 (m, 6H), 1.99 (dd, J=12.2, 5.6 Hz, 2H), 1.76-1.59 (m, 2H),1.57 (d, J=6.5 Hz, 6H), 1.52-1.40 (m, 2H), 1.30 (dd, J=9.7, 6.7 Hz, 2H),1.22 (d, J=6.8 Hz, 6H), 1.06 (d, J=6.0 Hz, 6H), 0.97 (app q, J=12.0 Hz,2H). LC-MS retention time 3.84 min; m/z 905.38 [M−H]⁻. LC data wasrecorded on a Shimadzu LC-10AS liquid chromatograph equipped with aPhenomenex-Luna 3u C18 2.0×50 mm column using a SPD-10AV UV-Vis detectorat a detector wave length of 220 nM. The elution conditions employed aflow rate of 0.8 mL/min, a gradient of 100% solvent A/0% solvent B to 0%solvent A/100% solvent B, a gradient time of 4 min, a hold time of 1min, and an analysis time of 5 min where solvent A was 5% MeOH/95%H₂O/10 mM ammonium acetate and solvent B was 5% H₂O/95% MeOH/10 mMammonium acetate. MS data was determined using a Micromass Platform forLC in electrospray mode.

Alternatively, Example 2 could be prepared as follows:

A mixture of(S)-2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(methoxycarbonylamino)aceticacid (Cap-2) (25.1 g, 91.0 mmol), 2-hydroxypyridine 1-oxide (10.12 g, 91mmol) and EDC (19.0 g, 99.0 mmol) in DMSO (400 mL) was stirred at rt for1 h. To this solution was added Example 1, step d (HCl salt; 29.2 g,41.4 mmol) and the resulting yellow solution was cooled to 10° C. with awater/ice bath. Then i-Pr₂EtN (26.7 g, 207 mmol) was added to thereaction mixture and, after the addition was complete, the ice bath wasremoved. The reaction mixture was stirred at ambient temperature for25.5 h before being quenched by pouring into an ice (1600 g) and water(400 mL) mixture. The mixture was stirred at ambient temperature for 3h. The white solid was collected on a Buchner funnel through filtrationand then dried under house vacuum with a stream of nitrogen passingthrough the top of the filter funnel overnight to give a wet solid (124g). The wet solid was dissolved into DCM (500 mL), washed with water(3×250 mL), dried (MgSO₄), filtered and concentrated to give a lightbrown solid (39.5 g). This material was combined with similar materialfrom another batch (total 44.4 g) and purified by chromatography usingan ISCO device (2×330 g silica gel, 0-30% MeOH/DCM) to afford Example-2(38.1 g) as a brown solid with an HPLC purity of 98.6%. The material wasdecolorized as follows: 43.1 g of material was dissolved in EtOH (500mL) and then treated with activated charcoal (8.6 g). The mixture washeated at 50° C. for 1 h, the charcoal was removed by filtration (3layers of filter paper) and the filter cake chased with additional EtOH(300 mL). The filtrate was evaporated to dryness and dried in vacuo witha warm water bath to afford an off-white solid (43.0 g). The productcould be purified further by employing an SFC protocol:

SFC Purification Conditions:

-   -   Throughput: 1.2 g/hr    -   Amt per Injections 30 mg    -   Column: Princeton CN 25×3 cm, 5μ    -   Column Temperature: 45° C.    -   Flow rate: 200 mL/min    -   Mobile Phase: CO₂/[MeOH/DCM=1:1 (in v/v)]=80/20    -   Injection Volume: 2.5 mL (12 mg/mL)    -   Injection Model: Stacked (injection/run time (min)=1.5/3.2)    -   Detector Wavelength: 316 nm    -   Sample Solvent: CH₃OH/DCM=1:1 (v/v)

EXAMPLE 3

Dimethyl((3-methyl-4,4′-biphenyldiyl)bis(1H-imidazole-4,2-diyl((2S,5S)-5-methyl-2,1-pyrrolidinediyl)((1S)-1-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-oxo-2,1-ethanediyl))biscarbamateExample 3, Step a

To a solution of 4-bromo-2-methylbenzoic acid (10 g, 46.5 mmol) in DMF(150 mL) was added N,O-dimethylhydroxylamine hydrochloride (5.44 g, 55.8mmol) at RT followed by HOBT (8.55 g, 55.8 mmol). Then EDC (10.7 g, 55.8mmol) was added followed by DIPEA (24.4 mL, 140 mmol) and the reactionmixture was stirred at RT for 12 h. The reaction mixture was thendiluted with EtOAc (150 mL), washed with water (3×250 mL) and brine (150mL), dried over Na₂SO₄, filtered and concentrated in vacuo to affordcrude Example 3, step a (9.5 g), which was submitted to the next step assuch. ¹H NMR (CDCl₃, δ=7.26 ppm, 400 MHz): δ 7.37 (d, J=1.6 Hz, 1H),7.34 (dd, J=8.0, 1.6 Hz, 1H), 7.14 (d, J=8.0 Hz, 1H), 3.47 (s, 3H), 3.30(s, 3H), 2.31 (s, 3H). LC/MS: Anal. Calcd. For [M+H]⁺ C₁₀H₁₃ ⁸¹BrNO₂:260.01. found 260.0.

Example 3, Step b

Example 3, step a (9.5 g, 36.8 mmol) was dissolved in diethyl ether (150mL) and cooled to 0° C. Then methylmagnesium iodide (3.0 M in diethylether, 24.54 mL, 73.6 mmol) was added drop wise over 10 minutes. Thereaction was stirred for 6 h at 40° C. and then brought to roomtemperature and stirred for 12 h. The reaction mixture was cooled to 0°C., quenched with ice and then with 1.5N HCl (50 mL). The organic layerwas separated and the aqueous layer was extracted with methyl tert-butylether (2×100 mL), dried over Na₂SO₄ and concentrated under reducedpressure. The crude was purified by flash chromatography (Silica gel,60-120, EtOAc: petroleum ether, 2:98) to afford Example 3, step b (6.25g) as pale yellow liquid. ¹H NMR (CDCl₃, δ=7.26 ppm, 400 MHz): δ 7.55(d, J=8.0 Hz, 1H), 7.41 (s, 1H), 7.40 (d, J=8.0 Hz, 1H), 2.55 (s, 3H),2.50 (s, 3H).

Example 3, Step c

4-Acetylphenylboronic acid (5.39 g, 32.9 mmol) was added to a sealedtube containing Example 3, step b (7.0 g, 32.9 mmol) in MeOH (75.0 mL)and the reaction mixture was purged with nitrogen for 10 minutes. ThenK₂CO₃ (9.08 g, 65.7 mmol) was added followed by Pd(Ph₃P)₄ (1.139 g,0.986 mmol) and the reaction mixture was purged with nitrogen forfurther 10 minutes. The reaction mixture was heated to 75° C. for 12 h.Then the reaction mixture was concentrated under reduced pressure andthe residue was diluted with EtOAc (100 mL) and washed with water (2×100mL), brine (50 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure. The residue was purified by combiflash (Silicycle,SiO₂, 10-15% EtOAc/petroleum ether) to afford Example 3, step c (6.5 g)as a white solid. ¹H NMR (CDCl₃, δ=7.26 ppm, 400 MHz): δ 8.06-8.04 (m,2H), 7.81 (d, J=8.0 Hz, 1H), 7.72-7.70 (m, 2H), 7.54-7.49 (m, 2H), 2.65(s, 3H), 2.63 (s, 6H).

Example 3, Step d

Bromine (1.12 mL, 21.8 mmol) (diluted in 10 mL of dioxane) was addedslowly (over 10 minutes) to a solution of Example 3, step c (2.75 g,10.90 mmol) in dioxane (50 mL) at 10° C., and the mixture was stirred atRT for 2 h. The reaction was quenched with 10% NaHCO₃ (25 mL) andextracted with DCM (50 mL). The organic phase was dried over Na₂SO₄ andconcentrated under reduced pressure to afford crude Example 3, step d(5.0 g) which was used as such in the next step without purification.LC/MS: Anal. Calcd. For [M+H]⁺ C₁₇H₁₅ ^(79/81)Br₂O₂: 410.94. found411.0.

Example 3, Step e

To a solution of crude Example 3, step d (5.1 g, 12 mmol) inacetonitrile (75 mL) was added(2S,5S)-1-(tert-butoxycarbonyl)-5-methylpyrrolidine-2-carboxylic acid(Example 1, step a) (5.70 g, 24.9 mmol) followed by DIPEA (8.69 mL, 49.7mmol) at 0° C. After 10 minutes, the temperature was raised to RT andstirred for 2 h. Then the reaction mixture was diluted with EtOAc (100mL) and washed with 10% NaHCO₃ (50 mL), brine (50 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure. The residuewas purified by combiflash (Silicycle, SiO₂, 25-30% EtOAc/petroleumether) to afford Example 3, step e (5.8 g) as a pale yellow oil. ¹H NMR(CDCl₃, δ=7.26 ppm, 400 MHz): δ 8.00 (app bd, 2H), 7.71 (app d, 3H),7.53-7.51 (m, 2H), 5.61-5.34 (m, 2H), 5.29-5.04 (m, 2H), 4.51-4.36 (m,2H), 4.09-3.91 (m, 2H), 2.59 (s, 3H), 2.35-2.21 (m, 4H), 2.15-2.04 (m,2H), 1.80-1.63 (m, 2H), 1.47/1.44 (s, 18H), 1.35-1.27 (m, 6H). LC/MS:Anal. Calcd. for [M−H]⁻ C₃₉H₄₉N₂O₁₀: 705.35. found 705.30.

Example 3, Step f

To a solution of Example 3, step e (5.6 g, 7.92 mmol) in xylenes (75 mL)was added NH₄OAc (12.21 g, 158 mmol), and the reaction mixture waspurged with nitrogen for 10 minutes. After heating for 18 h at 130° C.,the reaction mixture was cooled to room temperature and the volatilecomponents were removed under reduced pressure. Then the reactionmixture was diluted with EtOAc (100 mL) and washed with 10% NaHCO₃ (50mL), brine (50 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure. The residue was purified by combiflash (Redi Sep, C-18column, 30-40% acetonitrile:10 mM ammonium bicarbonate) to affordExample 3, step f (2.3 g) as pale yellow solid. ¹H NMR (DMSO-d₆, δ=2.50ppm, 400 MHz): δ 12.27/12.0/11.77/11.71 (s, 2H), 7.92-7.63 (m, 5H),7.58-7.47 (m, 3H), 7.24 (br s, 1H), 4.90-4.75 (m, 2H), 3.92-3.84 (m,2H), 2.54 (s, 3H), 2.20-2.01 (m, 6H), 1.73-1.65 (m, 2H), 1.48-1.12 (m,24H). LC/MS: Anal. Calcd. For [M−H]⁻ C₃₉H₄₉N₆O₄: 665.39. found 665.4.

Example 3, Step g

To a solution of Example 3, step g (1.55 g, 2.32 mmol) in MeOH (10 mL)was added in HCl/MeOH (4N, 58.1 mL) and stirred at room temperature for2 h. The volatile components were removed in vacuo, and the residue wasco-evaporated with dry DCM (3×25 mL). The resulting solid was exposed tohigh vacuum to afford the HCl salt of Example 3, step g (1.3 g) as apale yellow solid. ¹H NMR (MeOD, δ=3.34 ppm, 400 MHz): δ 8.06 (br s,1H), 7.98 (d, J=8.4 Hz, 2H), 7.90 (d, J=8.4 Hz, 2H), 7.86 (br s, 1H),7.78 (br s, 1H), 7.74 (d, J=8.4 Hz, 1H), 7.71 (d, J=8.4 Hz, 1H),5.27-5.20 (m, 2H), 4.04-4.00 (m, 2H), 2.80-2.67 (m, 4H), 2.59 (s, 3H),2.55-2.46 (m, 2H), 2.15-2.06 (m, 2H), 1.60 (d, J=6.4, 6H). LC/MS: Anal.Calcd. For [[M+H]⁺ C₂₉H₃₅N₆: 467.28. found 467.2.

EXAMPLE 3

HATU (60.5 mg, 0.159 mmol) was added to a stirred solution of(S)-2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-(methoxycarbonylamino)aceticacid (Cap-2) (39 mg, 0.16 mmol) and the HCl salt of Example 3, step g(44.3 mg, 0.072 mmol) in DMF (0.9 mL) and DIPEA (0.10 mL, 0.58 mmol).The reaction mixture was stirred at RT for 2 h and concentrated under astream of nitrogen overnight. The residue was diluted with MeOH,filtered and purified by prep HPLC (Phenomenex Luna C18(2) 100×30 mm, 10micron; MeOH/water w/ TFA buffer) to afford the TFA salt of dimethyl((3-methyl-4,4′-biphenyldiyl)bis(1H-imidazole-4,2-diyl((2S,5S)-5-methyl-2,1-pyrrolidinediyl)((1S)-1-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-oxo-2,1-ethanediyl)))biscarbamate(Example 3) (60.2 mg) as an off-white solid. ¹H NMR (400 MHz, MeOH-d₄) δ8.01-7.84 (m, 5H), 7.78-7.54 (m, 4H), 5.75 (d, J=6.0 Hz, 0.4H), 5.19(ddd, J=10.7, 6.9, 4.0 Hz, 1.6H), 4.84-4.72 (m, 2H), 4.36-4.12 (m, 4H),3.85-3.58 (m, 8H), 2.69-1.95 (m, 13H), 1.75-1.40 (m, 10H), 1.37-1.02 (m,14H), 0.96 (app q, J=12.2 Hz, 2H). LC-MS retention time 4.050 min; m/z461.31 [½M+H]⁺. LC data was recorded on a Shimadzu LC-10AS liquidchromatograph equipped with a Phenomenex-Luna 3u C18 2.0×50 mm columnusing a SPD-10AV UV-Vis detector at a detector wave length of 220 nM.The elution conditions employed a flow rate of 0.8 mL/min, a gradient of100% solvent A/0% solvent B to 0% solvent A/100% solvent B, a gradienttime of 4 min, a hold time of 1 min, and an analysis time of 5 min wheresolvent A was 5% MeOH/95% water/10 mM ammonium acetate and solvent B was5% water/95% MeOH/10 mM ammonium acetate. MS data was determined using aMicromass Platform for LC in electrospray mode.

EXAMPLE 4

Dimethyl((3-fluoro-4,4′-biphenyldiyl)bis(1H-imidazole-4,2-diyl((2S,5S)-5-methyl-2,1-pyrrolidinediyl)((1S)-1-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-oxo-2,1-ethanediyl))biscarbamateExample 4, Step a

To a solution of 1-(4-bromo-2-fluorophenyl)ethanone (5.0 g, 23 mmol) indioxane (150 mL) and ether (150 mL) in a ice-water bath at 0° C. wasadded bromine (1.18 mL, 23.0 mmol) dropwise. The reaction was stirredfor 1 hr, allowed to warm to RT and stirred for 16 hrs. The mixture waspartitioned between EtOAc (50 mL) and sat. NaHCO₃ (50 mL), and theorganic layer was washed with water and dried over Na₂SO₄. The volatilecomponent was evaporated in vacuo and the solid was dried under vacuumovernight to afford Example 4, step a (6.94 g) as white solid. ¹H NMR(DMSO-d₆, δ=2.5 ppm, 400 MHz): 7.87-7.79 (m, 2H), 7.62-7.60 (m, 1H),4.84 (s, 2H).

Example 4, Step b

To a solution of Example 4, step a (2.58 g, 8.72 mmol) and(2S,5S)-1-(tert-butoxycarbonyl)-5-methylpyrrolidine-2-carboxylic acid(2.00 g, 8.72 mmol) in acetonitrile (50 mL) was added DIEA (2.285 mL,13.08 mmol), and the mixture was stirred at room temperature for 64 hrs.Solvent was removed in vacuo and the residue was partitioned betweenEtOAc (40 mL) and water (30 mL). The organic layer was washed with sat.NaHCO₃ and brine, dried with Na₂SO₄ and evaporated in vacuo to affordExample 4, step b (3.8 g) as yellow solid. ¹H NMR (CDCl₃, 400 MHz): 7.87(m, 1H), 7.44 (m, 2H), 5.42-5.09 (m, 2H), 4.53-4.40 (m, 1H), 4.10-3.95(m, 1H), 2.31 (m, 2H), 2.09 (m, 1H), 1.75 (m, 1H), 1.49-1.46 (twosinglet, 9H), 1.33 (m, 3H). LC/MS: Anal. Calcd. for [M+Na]⁺C₁₉H₂₄BrNNaO₅: 466.06. found: 466.03.

Example 4, Step c

To a pressure tube containing a solution of Example 4, step b (3.8 g,8.6 mmol) in xylenes (40 mL) was added ammonium acetate (6.59 g, 86mmol), and the reaction vessel was capped and heated at 140° C. for 6hrs. The volatile component was evaporated in vacuo and the residue waspartitioned between DCM (80 mL) and water (50 mL). The organic layer wasseparated and washed with sat. NaHCO₃, and dried with Na₂SO₄. Removal ofthe solvent in vacuo resulted in a red oil which was purified by flashchromatograph (0-40% EtOAc/Hexane) to afford Example 4, step c (2.3 g)as brown solid. ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): 7.98 (app. t,J=8.4 Hz, 1H), 7.65 (dd, J=11, 1.9 Hz, 1H), 7.45 (dd, J=8.3, 2, 1H),7.36 (m, 1H), 4.85 (m, 1H), 3.90 (m, 1H), 2.15-2.07 (m, 3H), 1.73 (m,1H), 1.40-1.17 (m, 12H). LC/MS: Anal. Calcd. for [M+Na]⁺ C₁₉H₂₃⁷⁹BrFN₃NaO₂: 446.09. found: 446.00.

Example 4, Step d

To a solution of 2-bromo-1-(4-bromophenyl)ethanone (2.425 g, 8.72 mmol)and (2S,5S)-1-(tert-butoxycarbonyl)-5-methylpyrrolidine-2-carboxylicacid (2 g, 8.72 mmol) in acetonitrile (50 mL) was added DIEA (1.524 mL,8.72 mmol), and the mixture was stirred at room temperature for 16 hrs.Solvent was removed in vacuo and the residue was partitioned betweenEtOAc (40 mL) and water (30 mL). The organic phase was washed with sat.NaHCO₃ and brine, and dried with Na₂SO₄. Removal of the volatilecomponent in vacuo afforded Example 4, step d (1.74 g) as light yellowsolid, which was used without further purification. ¹H NMR (DMSO-d₆,δ=2.5 ppm, 400 MHz): 7.95-7.90 (m, 2H), 7.81 (m, 1H), 7.79 (m, 1H),5.63-5.44 (m, 2H), 4.36 (m, 1H), 3.99 (m, 1H), 2.27 (m, 1H), 2.09 (m,2H), 1.63 (m, 1H), 1.41-1.37 (two singlet, 9H), 1.19 (m, 3H). LC/MS:Anal. Calcd. for [M+Na]⁺ C₁₉H₂₄BrNNaO₅: 448.07. found: 448.06.

Example 4, Step e

To a pressure tube containing a solution of Example 4, step d (3.4 g,8.0 mmol) in xylenes (40 mL) was added ammonium acetate (6.15 g, 80mmol), and the mixture was heated at 140° C. for 6 hrs. The volatilecomponent was removed in vacuo, the residue was partitioned carefullybetween DCM (60 mL) and sat. NaHCO₃ (30 mL), and the organic layer wasseparated and dried with Na₂SO₄. The solvent was removed in vacuo togive red solid, which was purified by flash chromatograph (5-50%EtOAc/Hexane) to afford Example 4, step e (2.65 g) as light brown solid.¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): 7.73-7.71 (m, 2H), 7.59-7.50 (m,3H), 4.80 (m, 1H), 3.89 (m, 1H), 2.10 (m, 3H), 1.71 (m, 1H), 1.40-1.17(m, 12H). LC/MS: Anal. Calcd. for [M+Na]⁺ C₁₉H₂₄BrN₃NaO₂: 428.09. found:428.07.

Example 4, Step f

To a solution of Example 4, step e (2.64 g, 6.50 mmol) and4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (3.30 g,13.0 mmol) in dioxane (40 mL) was added potassium acetate (1.594 g,16.24 mmol). The mixture was degassed by bubbling nitrogen for 10 min,Pd(Ph₃P)₄ (0.375 g, 0.325 mmol) was added and degassing was continuedfor an additional 15 min. The reaction vessel was then sealed and heatedat 80° C. for 16 hrs. The volatile component was evaporated in vacuo andthe residue was partitioned between DCM (100 mL) and half sat. NaHCO₃(50 mL). The organic layer was separated, dried with Na₂SO₄, andevaporated in vacuo to afford a crude red oil which was purified byflash chromatograph (10-90% EtOAc/hexanes). Example 4, step f (2.7 g)was obtained as yellow foam. ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): 7.77(d, J=8.3 Hz, 2H), 7.64-7.53 (m, 3H), 4.80 (m, 1H), 3.88 (m, 1H), 2.09(m, 3H), 1.73 (m, 1H), 1.43-1.08 (m, 24H). LC/MS: Anal. Calcd. for[M+H]⁺ C₂₅H₃₇BrBN₃O₄: 454.29. found: 454.23.

Example 4, Step g

To a pressure tube containing a solution of Example 4, step f (2.70 g,5.96 mmol) and Example 4, step c (2.30 g, 5.42 mmol) in DME (70 mL) wereadded water (17.50 mL) and sodium bicarbonate (2.27 g, 27.1 mmol). Themixture was degassed by bubbling nitrogen for 15 min and Pd(Ph₃P)₄(0.313 g, 0.271 mmol) was added and degassing was continued for anadditional 15 min. The reaction vessel was sealed and heated at 80° C.for 15 hrs. The solvent was evaporated in vacuo and the residue waspartitioned between DCM (100 mL) and water (50 mL). The organic layerwas separated, dried with Na₂SO₄ and the volatile component was removedin vacuo and the resultant red crude solid was purified by flashchromatograph (30-100% EtOAc/Hexane). Example 4, step g (1.95 g) wasobtained as yellow solid. ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz): 8.10 (m,1H), 7.87-7.71 (m, 4H), 7.61-7.55 (m, 3H), 7.37 (m, 1H), 4.85 (m, 2H),3.91 (m, 2H), 2.11 (m, 6H), 1.76 (m, 2H), 1.42-1.08 (m, 24H). LC/MS:Anal. Calcd. for [M+H]⁺ C₃₅H₄₈FN₆O₄: 671.37. found: 671.35.

Example 4, Step h

To a suspension of Example 4, step g (1.95 g, 2.91 mmol) in dioxane (10mL) was added 4N HCl in dioxane (9.72 mL, 320 mmol), and the mixture wasstirred at room temperature for 6 hrs. Methanol (1 mL) was added andstirring was continued for 1 hr. The volatile component was removed invacuo and the residue was dried under vacuum overnight. The HCl salt ofExample 4, step h (1.7 g) was retrieved as yellow solid. ¹H NMR(DMSO-d₆, δ=2.5 ppm, 400 MHz): 10.34/10.29/9.43/9.08 (four broad S,˜4H), 8.16 (t, J=8.3 Hz, 1H), 8.10 (br s, 1H), 8.00 (d, J=8.3 Hz, 2H),7.92 (d, J=8.3 Hz, 2H), 7.78-7.72 (m, 3H), 4.99-4.89 (m, 2H), 3.80 (m,2H), 2.53-2.42 (m, 4H), 2.25 (m, 2H), 1.87 (m, 2H), 1.44 (d, J=6.5, 3H),1.43 (d, J=6.5, 3H). LC/MS: Anal. Calcd. for [M+H]⁺ C₂₈H₃₂FN₆: 471.27.found: 471.17.

EXAMPLE 4

HATU (59.4 mg, 0.156 mmol) was added to a stirred solution of(S)-2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-((methoxycarbonyl)amino)aceticacid (Cap-2) (38.3 mg, 0.156 mmol) and the HCl salt of Example 4, step h(43.8 mg, 0.071 mmol) in DMF (0.9 mL) and DIPEA (0.10 mL, 0.57 mmol).The reaction solution was stirred at RT for 2 h and concentrated under astream of nitrogen overnight. The residue was dissolved into MeOH (3mL), filtered and purified by prep HPLC (Phenomenex Luna C18(2) 100×30mm, 10 micron; MeOH/water w/ TFA buffer) to afford the TFA salt ofdimethyl((3-fluoro-4,4′-biphenyldiyl)bis(1H-imidazole-4,2-diyl((2S,5S)-5-methyl-2,1-pyrrolidinediyl)((1S)-1-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-oxo-2,1-ethanediyl)))biscarbamate(Example 4) (67.2 mg) as an off-white solid. ¹H NMR (400 MHz, MeOH-d₄) δ8.10-7.84 (m, 7H), 7.77-7.69 (m, 2H), 5.78-5.71 (m, 0.4H), 5.19 (td,J=10.6, 7.2 Hz, 1.6H), 4.83-4.74 (m, 2H), 4.36-4.12 (m, 4H), 3.85-3.62(m, 8H), 2.72-1.95 (m, 10H), 1.76-1.40 (m, 10H), 1.35-1.02 (m, 14H),0.96 (app q, J=12.2 Hz, 2H). LC-MS retention time 4.031 min; m/z 463.28[½M+H]⁺. LC data was recorded on a Shimadzu LC-10AS liquid chromatographequipped with a Phenomenex-Luna 3u C18 2.0×50 mm column using a SPD-10AVUV-Vis detector at a detector wave length of 220 nM. The elutionconditions employed a flow rate of 0.8 mL/min, a gradient of 100%solvent A/0% solvent B to 0% solvent A/100% solvent B, a gradient timeof 4 min, a hold time of 1 min, and an analysis time of 5 min wheresolvent A was 5% MeOH/95% water/10 mM ammonium acetate and solvent B was5% water/95% MeOH/10 mM ammonium acetate. MS data was determined using aMicromass Platform for LC in electrospray mode.

EXAMPLE 5

Dimethyl(4,4′-biphenyldiylbis(1H-imidazole-4,2-diyl((1R,3S,5R)-5-methyl-2-azabicyclo[3.1.0]hexane-3,2-diyl)((1S)-1-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-oxo-2,1-ethanediyl))biscarbamateExample 5, Step a

The above ester was prepared as a diastereomeric mixture from(S)-1-tert-butyl 2-methyl 5-oxopyrrolidine-1,2-dicarboxylate accordingto the procedure described in Tetrahedon Letters, 2003, 3203-3205.

Example 5, Step b

To a cooled (−50° C.) toluene (45 mL) solution of Example 5, step a(4.75 g, 18.5 mmol) was added Superhydride (19.20 mL of 1M/THF, 19.20mmol) dropwise over 10 min. Hunig's base (13.6 mL, 78 mmol) was added,and stirred for 10 min; DMAP (0.122 g, 0.997 mmol) was added as a solid,stirred for 15 min; and, trifluoroacetic anhydride (2.98 mL, 21.1 mmol)was added dropwise over 15 min, the cooling bath was removed, andstirring was continued for 4 hr while allowing it to warm to roomtemperature. The reaction mixture was washed with water (50 mL), sat.NaCl (30 mL), and the organic phase was concentrated in vacuo. Theresulting crude material was purified with flash chromatography (8-60%EtOAc/Hexane) to afford Example 5, step b as yellow oil (2.85 g). ¹H NMR(CDCl₃, 400 MHz): 6.36 (s, 0.5H), 6.25 (s, 0.5H), 4.70-4.57 (m, 1H),3.78 (s, 3H), 2.96 (m, 1H), 2.54 (m, 1H), 1.70 (s, 3H), 1.50 (s, 4.5H),1.44 (s, 4.5H).

Example 5, Step c

Diethylzinc (1.1 M in toluene, 59.1 mL, 65.0 mmol) was added dropwiseover 20 min to a cooled (−23° C.) toluene (60 mL) solution of Example 5,step b (5.23 g, 21.7 mmol), and stirred for 10 min. Chloroiodomethane(9.44 mL, 130 mmol) was added dropwise over 10 min, and the reactionmixture was stirred at −21° C. for 16 hr. Sat. NaHCO₃ (60 mL) was addedto the reaction mixture, the cooling bath was removed, and the mixturewas stirred for 10 min. It was then filtered, and the filter cake waswashed with toluene (50 mL). The filtrate was partitioned, and theorganic layer was dried with Na₂SO₄, and concentrated in vacuo. Theresulting crude material was purified with flash chromatography (2-10%EtOAc/Hexane) to afford Example 5, step c.1 (first elute; colorless oil;2.88 g) and Example 5, step c.2 (second elute; colorless oil; 1.01 g).Relative stereochemical assignment was made based on NOE studies.Example 5, step c.1: ¹H NMR (CDCl₃, 400 MHz): 4.65-4.52 (m, 1H), 3.72(s, 3H), 3.28-3.17 (m, 1H), 2.44-2.32 (m, 1H), 2.16-2.10 (m, 1H),1.51-1.42 (two s, 9H), 1.24 (s, 3H), 1.07 (m, 1H), 0.69-0.60 (m, 1H).Example 5, step c.2: ¹H NMR (CDCl₃, 400 MHz): 4.0 (m, 1H), 3.76 (s, 3H),3.32-3.16 (m, 1H), 2.43 (m, 1H), 2.01 (m, 1H), 1.44 (s, 9H), 1.35 (s,3H), 0.76-0.66 (m, 2H).

Example 5, Step d

To a solution of Example 5, step c.1 (2.88 g, 11.3 mmol) in ethanol (20mL) was added a solution of LiOH (0.324 g, 13.5 mmol) in water (10.00mL), and the reaction mixture was stirred at room temperature for 6 hr.Most of the volatile component was removed in vacuo, and the residue waspartitioned between water (20 mL) and ether (20 mL). The aqueous layerwas chilled in an ice-water bath, acidified with a 1N HCl to a pH regionof 2, and extracted with EtOAc (30 mL, 4×). The combined organic phasewas dried with Na₂SO₄ and evaporated in vacuo to afford Example 5, stepd.1 as a sticky solid (2.55 g). ¹H NMR (CDCl₃, 400 MHz): 4.64 (m, 1H),3.25 (appt s, 1H), 2.70-2.40 (m, 1H), 2.14 (m, 1H), 1.54-1.44 (m, 9H),1.27 (s, 3H), 1.10-0.80 (m, 1H), 0.67 (m, 1H). Example 5, step d.2 wasprepared similarly from Example 5, step c.2. ¹H NMR (CDCl₃, 400 MHz):4.13 (app br s, 1H), 3.06 (app br s, 1H), 2.55/2.41 (overlapping app brs, 2H), 1.51 (s, 9H), 1.27 (s, 3H), 0.76 (app t, J=5.6 Hz, 1H), 0.60(app br s, 1H).

Example 5, Step e

To a suspension of Example 5, step d.2 (1.09 g, 4.52 mmol) and1,1′-(biphenyl-4,4′-diyl)bis(2-bromoethanone) (0.869 g, 2.19 mmol) inacetonitrile (40 mL) was added DIEA (0.789 mL, 4.52 mmol), and themixture was stirred at room temperature for 4 hrs. The volatilecomponent was removed in vacuo, and the residue was partitioned betweenEtOAc (70 mL) and water (50 mL). The organic layer was washed with sat.NaHCO₃ (50 mL), dried with Na₂SO₄, evaporated in vacuo and dried undervacuum to give Example 5, step e (1.54 g) as white foam. ¹H NMR(DMSO-d₆, δ=2.5 ppm, 400 MHz): 8.13 (d, J=8.3 Hz, 4H), 7.99 (d, J=8.5Hz, 4H), 5.70-5.54 (m, 4H), 4.17 (m, 2H), 3.13-3.11 (m, 2H), 2.58-2.46(m, 2H), 2.19 (m, 2H), 1.42-1.37 (two s, 18H), 1.24 (s, 6H), 0.76-0.70(m, 4H). LC/MS: Anal. Calcd. for [M+Na]⁺ C₄₀H₄₈N₂NaO₁₀: 739.32. found:739.52.

Example 5, Step f

To a pressure tube containing a solution of Example 5, step e (1.54 g,2.15 mmol) in xylenes (40 mL) was added ammonium acetate (1.656 g, 21.48mmol), and the vessel was capped and heated at 140° C. for 5 hrs. Thevolatile component was removed in vacuo and the residue was carefullypartitioned between DCM (50 mL) and water (50 mL) while addingsufficient saturated NaHCO₃ solution so that at the end of partitioning,the aqueous phase is neutral or basic. The organic layer was dried withNa₂SO₄, evaporated in vacuo, and the resulting crude material waspurified by flash chromatograph (10-100% EtOAc/Hexane) to afford Example5, step f (0.65 g) as brown solid. ¹H NMR (DMSO-d₆, δ=2.5 ppm, 400 MHz):7.84-7.65 (m, 8H), 7.55-7.54 (m, 1.7H), 7.32-7.30 (m, 0.3H), 4.60 (m,2H), 3.20 (m, 2H), 2.48-2.43 (m, 2H), 2.12 (m, 2H), 1.45-1.07 (m, 24H),0.77 (m, 2H), 0.69 (m, 2H). LC/MS: Anal. Calcd. for [M+H]⁺ C₄₀H₄₉N₆O₄:677.38. found: 677.45.

Example 5, Step g

To a solution of Example 5, step f (0.65 g, 0.960 mmol) in dioxane (5mL) was added 4N HCl in dioxane (5.84 mL, 192 mmol), and the mixture wasstirred at room temperature for 6 hrs. The volatile component wasremoved in vacuo and dried under vacuum overnight to afford the HCl saltof Example 5, step g (0.6 g) as brown solid. ¹H NMR (DMSO-d₆, δ=2.5 ppm,400 MHz): 10.5-10 (br s, ˜3.2H), 7.99 (br s, 2H), 7.95 (d, J=8.5, 4H),7.85 (d, J=8.5 Hz, 4H), 4.76 (m, 2H), 3.18 (m, 2H), 2.61-2.46 (m, 4H;overlapped with solvent signal), 1.35 (s, 6H), 1.30 (m, 2H), 0.82 (appbr t, J=7.1, 2H). LC/MS: Anal. Calcd. for [M+H]⁺ C₃₀H₃₃N₆: 477.28.found: 477.22.

EXAMPLE 5

HATU (68.2 mg, 0.179 mmol) was added to a stirred solution of(S)-2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-((methoxycarbonyl)amino)aceticacid (Cap-2) (44 mg, 0.18 mmol) and the HCl salt of Example 5, step g(50.8 mg, 0.083 mmol) in DMF (0.8 mL) and DIPEA (0.12 mL, 0.67 mmol).The reaction solution was stirred at RT for 3 h and concentrated under astream of nitrogen overnight. The residue was dissolved into MeOH (˜2.5mL), filtered and purified by prep HPLC (Phenomenex Luna C18(2) 100×30mm, 10 micron; MeOH/water w/ TFA buffer) to afford the TFA salt ofdimethyl(4,4′-biphenyldiylbis(1H-imidazole-4,2-diyl((1R,3S,5R)-5-methyl-2-azabicyclo[3.1.0]hexane-3,2-diyl)((1S)-1-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-oxo-2,1-ethanediyl)))biscarbamate(Example 5) (49.5 mg) as yellow solid. ¹H NMR (400 MHz, MeOH-d₄) δ 7.93(s, 2H), 7.90-7.82 (m, 8H), 5.02 (t, J=8.5 Hz, 2H), 4.48 (d, J=8.0 Hz,2H), 4.26-4.18 (m, 2H), 3.80-3.71 (m, 2H), 3.68 (s, 6H), 3.60 (d, J=3.3Hz, 2H), 2.77 (dd, J=13.4, 9.2 Hz, 2H), 2.28 (dd, J=13.1, 7.8 Hz, 4H),1.56-1.44 (m, 6H), 1.42 (s, 6H), 1.25 (d, J=6.8 Hz, 6H), 1.09 (d, J=6.0Hz, 6H), 1.06-0.91 (m, 6H). LC-MS retention time 3.900 min; m/z 466.29[½M+H]⁺. LC data was recorded on a Shimadzu LC-10AS liquid chromatographequipped with a Phenomenex-Luna 3u C18 2.0×50 mm column using a SPD-10AVUV-Vis detector at a detector wave length of 220 nM. The elutionconditions employed a flow rate of 0.8 mL/min, a gradient of 100%solvent A/0% solvent B to 0% solvent A/100% solvent B, a gradient timeof 4 min, a hold time of 1 min, and an analysis time of 5 min wheresolvent A was 5% MeOH/95% water/10 mM ammonium acetate and solvent B was5% water/95% MeOH/10 mM ammonium acetate. MS data was determined using aMicromass Platform for LC in electrospray mode.

EXAMPLE 6

Dimethyl(4,4′-biphenyldiylbis(1H-imidazole-4,2-diyl(2S)-2,1-pyrrolidinediyl((1S)-1-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-oxo-2,1-ethanediyl))biscarbamate

HATU (81 mg, 0.212 mmol) was added to a stirred solution of the HCl saltof 4,4′-bis(2-((S)-pyrrolidin-2-yl)-1H-imidazol-5-yl)-1,1′-biphenyl(prepared in WO2008/021927) (56.2 mg, 0.099 mmol) and(S)-2-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-((methoxycarbonyl)amino)aceticacid (Cap-2) (52 mg, 0.21 mmol) in DMF (1.0 mL) and DIPEA (0.14 mL, 0.79mmol). The reaction solution was stirred at RT for 3 h and thenconcentrated under a stream of nitrogen. The residue was dissolved intoMeOH (˜5 mL), filtered and purified by prep HPLC (Phenomenex Luna C18(2)100×30 mm, 10 micron; MeOH/water w/ TFA buffer) to afford the TFA saltof dimethyl(4,4′-biphenyldiylbis(1H-imidazole-4,2-diyl(2S)-2,1-pyrrolidinediyl((1S)-1-((2R,6R)-2,6-dimethyltetrahydro-2H-pyran-4-yl)-2-oxo-2,1-ethanediyl)))biscarbamate(Example 6) (83 mg) as a light yellow solid. ¹H NMR (400 MHz, MeOH-d₄) δ7.96 (s, 2H), 7.91-7.84 (m, 8H), 5.26 (t, J=7.5 Hz, 2H), 4.24-4.09 (m,6H), 3.93-3.84 (m, 2H), 3.74-3.68 (m, 2H), 3.67 (s, 6H), 2.63-2.55 (m,2H), 2.39-2.12 (m, 8H), 1.57 (d, J=11.0 Hz, 2H), 1.51-1.41 (m, 2H),1.32-1.25 (m, 2H), 1.22 (d, J=7.0 Hz, 6H), 1.05 (d, J=6.0 Hz, 6H), 0.98(app q, J=12.3 Hz, 2H). LC-MS retention time 1.660 min; m/z 879.8[M+H]⁺. LC data was recorded on a Shimadzu LC-10AS liquid chromatographequipped with a Phenomenex-Luna 3u C18 2.0×50 mm column using a SPD-10AVUV-Vis detector at a detector wave length of 220 nM. The elutionconditions employed a flow rate of 0.8 mL/min, a gradient of 100%solvent A/0% solvent B to 0% solvent A/100% solvent B, a gradient timeof 4 min, a hold time of 1 min, and an analysis time of 5 min wheresolvent A was 10% Acetonitrile/90% water/0.1% trifluoroacetic acid andsolvent B was 10% water/90% Acetonitrile/0.1% trifluoroacetic acid. MSdata was determined using a Micromass Platform for LC in electrospraymode.

Biological Activity

An HCV Replicon assay was utilized in the present disclosure, and wasprepared, conducted and validated as described in commonly ownedPCT/US2006/022197 and in O'Boyle et. al. Antimicrob Agents Chemother.2005 April; 49(4):1346-53. Recommended assay methods incorporatingluciferase reporters have also been used as described from commercialsources (Apath.com).

HCV-neo replicon cells and replicon cells containing resistancesubstitutions in the NS5A region were used to test the currentlydescribed family of compounds. The compounds were determined to havediffering degrees of reduced inhibitory activity on cells containingmutations vs. the corresponding inhibitory potency against wild-typecells. Thus, the compounds of the present disclosure can be effective ininhibiting the function of the HCV NS5A protein and are understood to beas effective in combinations as previously described in applicationPCT/US2006/022197 and commonly owned WO/04014852. It should beunderstood that the compounds of the present disclosure can inhibitmultiple genotypes of HCV. Table 2 shows the EC₅₀ (Effective 50%inhibitory concentration) values of representative compounds of thepresent disclosure against the HCV genotype 1b wild type, HCV genotype1b LV/YH double resistant mutant, HCV genotype 1a, and genotype-1a YHsingle resistant mutant.

The compounds of the present disclosure may inhibit HCV by mechanisms inaddition to or other than NS5A inhibition. In one embodiment thecompounds of the present disclosure inhibit HCV replicon and in anotherembodiment the compounds of the present disclosure inhibit NS5A.Compounds of the present disclosure may inhibit multiple genotypes ofHCV containing multiple variants of NS5A sequences.

IV and PO Single Dose Pharmacokinetic Studies in Rat

The pharmacokinetics of Examples 1 to 6 and Compound-A (WO2008/021927)were characterized in male Sprague-Dawley rats (260-310 g) (see Table2). In these studies, two groups of animals (N=3 per group) receivedcompound either as an intravenous (IV) infusion (2 mg/kg over 10minutes) via the jugular vein or by oral gavage (5 mg/kg) in a vehicleof 100% PEG 400 or 90:5:5 PEG 400:ethanol:TPGS, respectively. The ratsin the oral dosing group were fasted overnight. Serial blood sampleswere obtained at 0.17 (IV only), 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 24h post dose. Blood samples (˜0.3 mL) were collected from the jugularvein into K₃EDTA-containing tubes and then centrifuged at 4° C.(1500-2000×g) to obtain plasma. Plasma samples were stored at −20° C.until analysis by LC/MS/MS.

A bioanalytical method utilizing liquid chromatography separationfollowed by tandem mass spectrometry detection (LC/MS/MS) was developedfor the compound analysis in rat plasma. Detection was performed usingselected reaction monitoring. Ions representing the precursor (M+H)⁺species were selected in quadrupole 1 and collisionally dissociated withN₂ to generate specific product ions, which were subsequently monitoredby quadrupole 3. Standard curves were prepared in male rat plasma andprocessed in the same manner as test samples to generate quantitativedata.

Pharmacokinetic parameter values were calculated using noncompartmentalmethods by Kinetica. Values below the lower limit of quantification(LLOQ) were not used in calculations. Area under the curve (AUC) wascalculated using the linear trapezoidal rule.

TABLE 2 24 h rat PK (Auc, nM · h; EC50 (uM) G 1a oral G 1b wild G 1a(CC50, bioavailability) type G 1b LV/YH wild type G 1a YH uM) Compound-A103; 1.3% 1.19E−04 0.020 5.11E−05 0.013 63.90 Example-1 1603; 15%3.96E−06 3.19E−03 5.48E−06 9.77E−04 8.46 Example-2 2219; 12% 8.03E−061.15E−04 6.43E−06 1.58E−04 5.89 Example-3 4486; 17% 6.41E−06 1.91E−045.20E−06 1.08E−03 6.15 Example-4 1729; 16% 4.15E−06 4.36E−04 5.95E−064.02E−04 7.75 Example-5 539; 6.3% 6.85E−06 9.85E−06 3.72E−06 6.07E−055.34 Example-6 1356; 5% 3.67E−05 6.59E−04 1.62E−05 3.83E−04 15.42

1. A compound of Formula (I)

or a pharmaceutically acceptable salt thereof, wherein R¹ is selectedfrom hydrogen, methyl, and fluoro; R² is selected from hydrogen andmethyl; R³ and R⁴ are each hydrogen; or R³ and R⁴, together with thecarbon atoms to which they are attached, form a cyclopropyl ring; and R⁵is selected from hydrogen and methyl.
 2. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R¹ is hydrogen.
 3. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R¹ is fluoro.
 4. The compound of claim 3, or a pharmaceuticallyacceptable salt thereof, wherein R², R³, and R⁴ are each hydrogen; andR⁵ is methyl.
 5. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein R¹ is methyl.
 6. The compound of claim5 wherein R², R³, and R⁴ are each hydrogen; and R⁵ is methyl.
 7. Acompound selected from

or a pharmaceutically acceptable salt thereof.
 8. A compound which is

or a pharmaceutically acceptable salt thereof.
 9. A compound which is

or a pharmaceutically acceptable salt thereof.
 10. A compositioncomprising a compound of claim 1, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.
 11. The compositionof claim 10 further comprising one, two, or three additional compoundshaving anti-HCV activity.
 12. The composition of claim 11 wherein atleast one of the additional compounds is an interferon or a ribavirin.13. The composition of claim 12 wherein the interferon is selected frominterferon alpha 2B, pegylated interferon alpha, consensus interferon,interferon alpha 2A, interferon lambda, and lymphoblastiod interferontau.
 14. The composition of claim 11 wherein at least one of theadditional compounds is selected from interleukin 2, interleukin 6,interleukin 12, a compound that enhances the development of a type 1helper T cell response, interfering RNA, anti-sense RNA, Imiquimod,ribavirin, an inosine 5′-monophospate dehydrogenase inhibitor,amantadine, and rimantadine.
 15. The composition of claim 11 wherein atleast one of the additional compounds is effective to inhibit thefunction of a target selected from HCV metalloprotease, HCV serineprotease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCVassembly, HCV egress, HCV NS5A protein, and IMPDH for the treatment ofan HCV infection.
 16. A method of treating an HCV infection in apatient, comprising administering to the patient a therapeuticallyeffective amount of a compound of claim 1, or a pharmaceuticallyacceptable salt thereof.
 17. The method of claim 16 further comprisingadministering one, two, or three additional compounds having anti-HCVactivity prior to, after or simultaneously with the compound of claim 1,or a pharmaceutically acceptable salt thereof.
 18. The method of claim17 wherein at least one of the additional compounds is an interferon ora ribavirin.
 19. The method of claim 18 wherein the interferon isselected from interferon alpha 2B, pegylated interferon alpha, consensusinterferon, interferon alpha 2A, interferon lambda, and lymphoblastiodinterferon tau.
 20. The method of claim 17 wherein at least one of theadditional compounds is selected from interleukin 2, interleukin 6,interleukin 12, a compound that enhances the development of a type 1helper T cell response, interfering RNA, anti-sense RNA, Imiquimod,ribavirin, an inosine 5′-monophospate dehydrogenase inhibitor,amantadine, and rimantadine.
 21. The method of claim 17 wherein at leastone of the additional compounds is effective to inhibit the function ofa target selected from HCV metalloprotease, HCV serine protease, HCVpolymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCVegress, HCV NS5A protein, and IMPDH for the treatment of an HCVinfection.